Methods for generating cardiomyocytes

ABSTRACT

The present disclosure provides method of generating cardiomyocytes from post-natal fibroblasts. The present disclosure further provides cells and compositions for use in generating cardiomyocytes.

CROSS-REFERENCE

This application is a divisional application of U.S. patent applicationSer. No. 13/642,391, filed Apr. 15, 2013, which is a national phasefiling under 35 U.S.C. §371 of PCT/US2011/033938, filed Apr. 26, 2011,which claims the benefit of U.S. Provisional Patent Application No.61/328,988, filed Apr. 28, 2010, and of U.S. Provisional PatentApplication No. 61/364,295, filed Jul. 14, 2010, which applications areincorporated herein by reference in their entirety.

BACKGROUND

Heart disease is a leading cause of adult and childhood mortality in thewestern world. The underlying pathology is typically loss ofcardiomyocytes that leads to heart failure, or improper development ofcardiomyocytes during embryogenesis that leads to congenital heartmalformations. Because cardiomyocytes have little or no regenerativecapacity, current therapeutic approaches are limited. Embryonic stemcells possess clear cardiogenic potential, but efficiency of cardiacdifferentiation, risk of tumor formation, and issues of cellularrejection must be overcome.

There is a need in the art for methods of generating cardiomyocytes.

LITERATURE

-   U.S. Patent Publication No. 2009/0208465; U.S. Pat. No. 7,682,828;    U.S. Patent Publication No. 2010/0075421; WO 2009/152484; WO    2009/152485; Takahashi and Yamanaka (2006) Cell 126:663.

SUMMARY OF THE INVENTION

The present disclosure provides method of generating cardiomyocytes frompost-natal fibroblasts. The present disclosure further provides cellsand compositions for use in generating cardiomyocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-I depict the results of screening for cardiomyocyte-inducingfactors.

FIGS. 2A-F depict the effect of various factors on cardiac geneexpression in fibroblasts.

FIGS. 3A-J depict reprogramming of cardiac fibroblasts directly intocardiomyocytes.

FIGS. 4A-D depict reprogramming of gene expression in inducedcardiomyocytes (iCMs).

FIGS. 5A-D depict spontaneous Ca²⁺ flux, electrical activity, andbeating in iCMs.

FIGS. 6A-C depict in vivo reprogramming of cardiac fibroblasts intocardiomyocytes.

FIGS. 7A-G depict in vivo reprogramming of cardiac fibroblasts tocardiomyocyte-like cells.

FIGS. 8A-N depict single-cell analysis of cardiac reprogramming in vivo.

FIG. 9 depicts intracellular recordings showing action potentials foradditional in vivo reprogrammed iCMs.

FIGS. 10A-D depict determination of area at risk (AAR) and infarct sizefor dsRed or GMT (expression vector encoding three factors:Gata4-Mef2c-Tbx-5) injected hearts after coronary ligation andechocardiography data.

FIGS. 11A-D depict the effect of in vivo delivery of cardiacreprogramming factors on cardiac function after myocardial infarction.

FIGS. 12A-F and FIGS. 13A-E depict the effect of thymosin β4 on cardiacfibroblasts upon injury and on in vivo reprogramming.

DEFINITIONS

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The term includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; and the like.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein and refer to a polymeric form of nucleotides of any length,either deoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides,vectors, probes, and primers.

The term “operably linked” refers to functional linkage betweenmolecules to provide a desired function. For example, “operably linked”in the context of nucleic acids refers to a functional linkage betweennucleic acids to provide a desired function such as transcription,translation, and the like, e.g., a functional linkage between a nucleicacid expression control sequence (such as a promoter, signal sequence,or array of transcription factor binding sites) and a secondpolynucleotide, wherein the expression control sequence affectstranscription and/or translation of the second polynucleotide.

As used herein the term “isolated” with reference to a cell, refers to acell that is in an environment different from that in which the cellnaturally occurs, e.g., where the cell naturally occurs in amulticellular organism, and the cell is removed from the multicellularorganism, the cell is “isolated.” An isolated genetically modified hostcell can be present in a mixed population of genetically modified hostcells, or in a mixed population comprising genetically modified hostcells and host cells that are not genetically modified. For example, anisolated genetically modified host cell can be present in a mixedpopulation of genetically modified host cells in vitro, or in a mixed invitro population comprising genetically modified host cells and hostcells that are not genetically modified.

A “host cell,” as used herein, denotes an in vivo or in vitro cell(e.g., a eukaryotic cell cultured as a unicellular entity), whicheukaryotic cell can be, or has been, used as recipients for a nucleicacid (e.g., an exogenous nucleic acid) or an exogenous polypeptide(s),and include the progeny of the original cell which has been modified byintroduction of the exogenous polypeptide(s) or genetically modified bythe nucleic acid. It is understood that the progeny of a single cell maynot necessarily be completely identical in morphology or in genomic ortotal DNA complement as the original parent, due to natural, accidental,or deliberate mutation.

The term “genetic modification” and refers to a permanent or transientgenetic change induced in a cell following introduction of new nucleicacid (i.e., nucleic acid exogenous to the cell). Genetic change(“modification”) can be accomplished by incorporation of the new nucleicacid into the genome of the host cell, or by transient or stablemaintenance of the new nucleic acid as an extrachromosomal element.Where the cell is a eukaryotic cell, a permanent genetic change can beachieved by introduction of the nucleic acid into the genome of thecell. Suitable methods of genetic modification include viral infection,transfection, conjugation, protoplast fusion, electroporation, particlegun technology, calcium phosphate precipitation, direct microinjection,and the like.

As used herein, the term “exogenous nucleic acid” refers to a nucleicacid that is not normally or naturally found in and/or produced by acell in nature, and/or that is introduced into the cell (e.g., byelectroporation, transfection, infection, lipofection, or any othermeans of introducing a nucleic acid into a cell).

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc. Insome embodiments, the individual is a human. In some embodiments, theindividual is a murine.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound or a number of cells that, when administered to amammal or other subject for treating a disease, is sufficient to effectsuch treatment for the disease. The “therapeutically effective amount”will vary depending on the compound or the cell, the disease and itsseverity and the age, weight, etc., of the subject to be treated.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aninduced cardiomyocyte” includes a plurality of such cardiomyocytes andreference to “the post-natal fibroblast” includes reference to one ormore post-natal fibroblasts and equivalents thereof known to thoseskilled in the art, and so forth. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides method of generating cardiomyocytes frompost-natal fibroblasts. The present disclosure further provides cellsand compositions for use in generating cardiomyocytes.

Methods of Generating Cardiomyocytes

The present disclosure provides method of generating cardiomyocytes frompost-natal fibroblasts. The methods generally involve introducing into apost-natal fibroblast one or more reprogramming factors. In some cases,the polypeptides themselves are introduced into a post-natal fibroblast.In other cases, the post-natal fibroblast is genetically modified withone or more nucleic acids comprising nucleotide sequences encoding there-programming factors.

In some embodiments, the methods involve introducing into a post-natalfibroblast one or more reprogramming factors selected from Gata4, Mef2c,Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf polypeptides. In somecases, the polypeptides themselves are introduced into a post-natalfibroblast. In other cases, the post-natal fibroblast is geneticallymodified with one or more nucleic acids comprising nucleotide sequencesencoding Gata4, Mef2c, and Tbx5 polypeptides.

In some embodiments, the methods involve introducing into a post-natalfibroblast three (and only three) reprogramming factors: Gata4, Mef2c,and Tbx5 polypeptides. In some cases, the polypeptides themselves areintroduced into a post-natal fibroblast. In other cases, the post-natalfibroblast is genetically modified with one or more nucleic acidscomprising nucleotide sequences encoding Gata4, Mef2c, and Tbx5polypeptides.

Cardiomyocytes generated directly from post-natal fibroblasts using asubject method are referred to herein as “induced cardiomyocytes.”Polypeptides such as Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd,Smyd1, and Srf are also referred to collectively herein as“reprogramming factors,” or “reprogramming transcription factors.” Apost-natal fibroblast into one or more reprogramming factors areintroduced is reprogrammed directly into a differentiated cardiomyocyte,without first becoming a stem cell or a progenitor cell.

As noted above, in some cases, a subject method of generating acardiomyocyte involves genetically modifying a post-natal fibroblastwith one or more nucleic acids comprising nucleotide sequences encodingone or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1,and Srf polypeptides. The reprogramming factors encoded by thenucleotide sequences are produced in the post-natal fibroblast and, as aresult of the production of the one or more reprogramming factors, thegenetically modified fibroblast is reprogrammed directly into adifferentiated cardiomyocyte. The genetically modified fibroblast isreprogrammed directly into a differentiated cardiomyocyte, without firstbecoming a stem cell or progenitor cell.

As noted above, in some cases, a subject method of generating acardiomyocyte involves genetically modifying a post-natal fibroblastwith one or more nucleic acids comprising nucleotide sequences encodingGata4, Mef2c, and Tbx5 polypeptides. The Gata4, Mef2c, and Tbx5polypeptides are produced in the post-natal fibroblast and, as a resultof the production of the Gata4, Mef2c, and Tbx5 polypeptides, thegenetically modified fibroblast is reprogrammed directly into adifferentiated cardiomyocyte. The genetically modified fibroblast isreprogrammed directly into a differentiated cardiomyocyte, without firstbecoming a stem cell or progenitor cell.

In some cases, a post-natal fibroblast is modified by introducing one ormore of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides themselves into a host post-natal fibroblast. A post-natalfibroblast into which one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf polypeptides has been introduced (either byintroducing the polypeptides themselves or by introducing one or morenucleic acids comprising nucleotide sequence encoding the one or morepolypeptides) is referred to as a “modified fibroblast” or a “modifiedpost-natal fibroblast.” A post-natal fibroblast into which one or morenucleic acids comprising nucleotide sequence encoding one or more ofGata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides has been introduced is referred to as a “geneticallymodified fibroblast” or a “genetically modified post-natal fibroblast.”

In some cases, a post-natal fibroblast is modified by introducing Gata4,Mef2c, and Tbx5 polypeptides themselves into a host post-natalfibroblast. A post-natal fibroblast into which Gata4, Mef2c, and Tbx5polypeptides have been introduced (either by introducing thepolypeptides themselves or by introducing one or more nucleic acidscomprising nucleotide sequence encoding Gata4, Mef2c, and Tbx5polypeptides) is referred to as a “modified fibroblast” or a “modifiedpost-natal fibroblast.” A post-natal fibroblast into which one or morenucleic acids comprising nucleotide sequence encoding Gata4, Mef2c, andTbx5 polypeptides have been introduced is referred to as a “geneticallymodified fibroblast” or a “genetically modified post-natal fibroblast.”

As noted above, using a subject method, one or more reprogrammingfactors (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1,Myocd, Smyd1, and Srf; or a subset of reprogramming factors comprisingGata4, Mef2c, and Tbx5 polypeptides) are introduced into a post-natalfibroblast (e.g., the reprogramming factor polypeptide(s) themselves areintroduced into a post-natal fibroblast; or a post-natal fibroblast isgenetically modified with one or more nucleic acids comprisingnucleotide sequences encoding reprogramming factor polypeptide(s)), andas a result, the modified fibroblast is reprogrammed directly into adifferentiated cardiomyocyte, without first becoming a stem cell orprogenitor cell. Thus, for example, the modified or genetically modifiedfibroblast does not produce detectable levels of an early cardiacprogenitor marker. For example, the modified or genetically modifiedfibroblast does not produce detectable levels of Isl1, an early cardiacprogenitor marker that is transiently expressed before cardiacdifferentiation.

In some embodiments, a post-natal fibroblast is genetically modified invitro with one or more nucleic acids comprising nucleotide sequencesencoding one or more reprogramming factors (e.g., one or more of Gata4,Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides);or one or more reprogramming factor polypeptides themselves (e.g., oneor more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, andSrf; or a subset of reprogramming factors comprising Gata4, Mef2c, andTbx5 polypeptides) are introduced in vitro into a post-natal fibroblast;where the modified or genetically modified fibroblasts becomecardiomyocytes in vitro. Once the fibroblasts are reprogrammed directlyinto cardiomyocytes in vitro, generating induced cardiomyocytes, theinduced cardiomyocytes can be introduced into an individual.

For example, in some embodiments, a subject method involves: a)genetically modifying a post-natal fibroblast in vitro with one or morenucleic acids comprising nucleotide sequences encoding one or more ofGata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides, where production of the encoded polypeptides in thegenetically modified fibroblasts results in reprogramming of thegenetically modified fibroblast directly into a cardiomyocyte in vitro,thereby generating an induced cardiomyocyte; and b) introducing theinduced cardiomyocyte(s) into an individual. In other embodiments, asubject method involves: a) introducing one or more of Gata4, Mef2c,Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf polypeptides into apost-natal fibroblast in vitro, generating a modified fibroblast, wherethe modified fibroblast, as a result of introduction of the one or moreof Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides, is reprogrammed directly into a cardiomyocyte in vitro,thereby generating an induced cardiomyocyte; and b) introducing theinduced cardiomyocyte into an individual.

As another example, in some embodiments, a subject method involves: a)genetically modifying a post-natal fibroblast in vitro with one or morenucleic acids comprising nucleotide sequences encoding Gata4, Mef2c, andTbx5 polypeptides, where production of the Gata4, Mef2c, and Tbx5polypeptides in the genetically modified fibroblasts results inreprogramming of the genetically modified fibroblast directly into acardiomyocyte in vitro, thereby generating an induced cardiomyocyte; andb) introducing the induced cardiomyocyte(s) into an individual. In otherembodiments, a subject method involves: a) introducing Gata4, Mef2c, andTbx5 polypeptides into a post-natal fibroblast in vitro, generating amodified fibroblast, where the modified fibroblast, as a result ofintroduction of the Gata4, Mef2c, and Tbx5 polypeptides, is reprogrammeddirectly into a cardiomyocyte in vitro, thereby generating an inducedcardiomyocyte; and b) introducing the induced cardiomyocyte into anindividual.

In other embodiments, a post-natal fibroblast is genetically modified invitro with one or more nucleic acids comprising nucleotide sequencesencoding one or more reprogramming factors (e.g., one or more of Gata4,Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides);or one or more reprogramming factor polypeptides themselves (e.g., oneor more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, andSrf; or a subset of reprogramming factors comprising Gata4, Mef2c, andTbx5 polypeptides) are introduced in vitro into a post-natal fibroblast;and the modified or genetically modified fibroblasts are introduced intoan individual, where the modified or genetically modified fibroblastsare reprogrammed directly into cardiomyocytes in vivo.

Thus, for example, in some embodiments, a subject method involves: a)genetically modifying a post-natal fibroblast in vitro with one or morenucleic acids comprising nucleotide sequences encoding one or more ofGata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf;polypeptides; and b) introducing the genetically modified fibroblastsinto an individual, where production of the one or more of Gata4, Mef2c,Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; polypeptides in thegenetically modified fibroblasts results in reprogramming of thegenetically modified fibroblast directly into a cardiomyocyte in vivo,thereby generating an induced cardiomyocyte in the individual. In otherembodiments, a subject method involves: a) introducing one or more ofGata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf;polypeptides into a post-natal fibroblast in vitro, generating amodified fibroblast; and b) introducing the modified fibroblast(s) intoan individual, where the modified fibroblasts, as a result ofintroduction of the one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf; polypeptides, are reprogrammed directlyinto a cardiomyocyte in vivo, thereby generating an inducedcardiomyocyte in the individual.

As another example, in some embodiments, a subject method involves: a)genetically modifying a post-natal fibroblast in vitro with one or morenucleic acids comprising nucleotide sequences encoding Gata4, Mef2c, andTbx5 polypeptides; and b) introducing the genetically modifiedfibroblasts into an individual, where production of the Gata4, Mef2c,and Tbx5 polypeptides in the genetically modified fibroblasts results inreprogramming of the genetically modified fibroblast directly into acardiomyocyte in vivo, thereby generating an induced cardiomyocyte inthe individual. In other embodiments, a subject method involves: a)introducing Gata4, Mef2c, and Tbx5 polypeptides into a post-natalfibroblast in vitro, generating a modified fibroblast; and b)introducing the modified fibroblast(s) into an individual, where themodified fibroblasts, as a result of introduction of the Gata4, Mef2c,and Tbx5 polypeptides, are reprogrammed directly into a cardiomyocyte invivo, thereby generating an induced cardiomyocyte in the individual.

In other embodiments, a post-natal fibroblast is genetically modified invivo with one or more nucleic acids comprising nucleotide sequencesencoding one or more reprogramming factors (e.g., one or more of Gata4,Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides),or one or more reprogramming factor polypeptides themselves (e.g., oneor more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, andSrf; or a subset of reprogramming factors comprising Gata4, Mef2c, andTbx5 polypeptides) are introduced in vivo into a post-natal fibroblast;and the modified or genetically modified fibroblasts are reprogrammeddirectly into cardiomyocytes in vivo.

A post-natal fibroblast that is genetically modified with one or morenucleic acids comprising nucleotide sequences encoding one or morereprogramming factors (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1,Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset of reprogrammingfactors comprising Gata4, Mef2c, and Tbx5 polypeptides), or that ismodified with one or more reprogramming factor polypeptides themselves(e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd,Smyd1, and Srf; or a subset of reprogramming factors comprising Gata4,Mef2c, and Tbx5 polypeptides), is reprogrammed into a differentiatedcardiomyocyte in a time period of from about 5 days to about 7 days, orfrom about 7 days to about 14 days. For example, where a population ofpost-natal fibroblasts is genetically modified or modified byintroducing reprogramming factor polypeptides, as described above, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 50%, at least about 75%, or morethan 75% (e.g., at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, or more than 98%), of thepopulation is reprogrammed into differentiated cardiomyocytes (inducedcardiomyocytes) in a time period of from about 5 days to about 7 days,from about 7 days to about 14 days, or from about 2 weeks to about 4weeks.

In some embodiments, where a population of post-natal fibroblasts isgenetically modified with one or more nucleic acids comprisingnucleotide sequences encoding one or more reprogramming factors (e.g.,one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1,and Srf; or a subset of reprogramming factors comprising Gata4, Mef2c,and Tbx5 polypeptides), or where a population of post-natal fibroblastsis modified by introducing one or more reprogramming factors themselves(e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd,Smyd1, and Srf; or a subset of reprogramming factors comprising Gata4,Mef2c, and Tbx5 polypeptides) into the fibroblasts, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 50%, at least about 75%, or more than 75%(e.g., at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or more than 98%), of thepopulation is cTnT⁺ (i.e., expresses cardiac troponin T) in a timeperiod of from about 5 days to about 7 days, from about 7 days to about14 days, or from about 2 weeks to about 4 weeks.

In some embodiments, a subject method of generating inducedcardiomyocytes involves genetically modifying a host post-natalfibroblast (or a population of host post-natal fibroblasts) with one ormore nucleic acids comprising nucleotide sequences encoding one or morereprogramming factors (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1,Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset of reprogrammingfactors comprising Gata4, Mef2c, and Tbx5 polypeptides); generating apopulation of genetically modified post-natal fibroblasts, and, after atime (e.g., 5 days to 7 days, 1 week to 2 weeks, or 2 weeks to 4 weeks),sorting the population of genetically modified post-natal fibroblasts toenrich for cardiomyocytes. The population of genetically modifiedpost-natal fibroblasts can be sorted for expression of afibroblast-specific marker, to remove any remaining fibroblasts. Thepopulation of genetically modified post-natal fibroblasts can be sortedfor expression of a cardiomyocyte-specific marker.

In some embodiments, a subject method of generating inducedcardiomyocytes involves modifying a host post-natal fibroblast (or apopulation of host post-natal fibroblasts) by introducing one or morereprogramming factor polypeptides (e.g., one or more of Gata4, Mef2c,Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides)into host post-natal fibroblasts; generating a population of modifiedpost-natal fibroblasts, and, after a time (e.g., 5 days to 7 days, 1week to 2 weeks, or 2 weeks to 4 weeks), sorting the population ofmodified post-natal fibroblasts to enrich for cardiomyocytes. Thepopulation of modified post-natal fibroblasts can be sorted forexpression of a fibroblast-specific marker, to remove any remainingfibroblasts. The population of modified post-natal fibroblasts can besorted for expression of a cardiomyocyte-specific marker.

In some embodiments, a host post-natal fibroblast is geneticallymodified with one or more nucleic acids comprising nucleotide sequencesencoding one or more reprogramming factors (e.g., one or more of Gata4,Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides),or is modified by introducing one or more reprogramming factorpolypeptides (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf; or a subset of reprogramming factorscomprising Gata4, Mef2c, and Tbx5 polypeptides) into host post-natalfibroblasts); and is also genetically modified with a nucleic acidcomprising a nucleotide sequence encoding a detectable marker (e.g., apolypeptide that directly produces a detectable signal; an enzyme thatproduces a detectable signal upon acting on a substrate), where thedetectable marker-encoding nucleotide sequence is operably linked to acardiomyocyte-specific promoter. Suitable polypeptides that provide adirect detectable signal include a fluorescent protein such as a greenfluorescent protein, a yellow fluorescent protein, a blue fluorescentprotein, etc. Suitable enzymes that produce a detectable signal uponacting on a substrate include, e.g., luciferase (acting on the substrateluciferin), alkaline phosphatase, and the like. Cardiomyocyte-specificpromoters include, e.g., an α-myosin heavy chain promoter; a cTnTpromoter; and the like. Expression of the detectable marker can providefor detection of an induced cardiomyocyte; and can provide a means ofsorting for induced cardiomyocytes.

The post-natal fibroblasts that serve as host cells for modification orgenetic modification, as described above, can be from any of a varietyof sources. Mammalian fibroblasts, e.g., human fibroblasts, murine(e.g., mouse) fibroblasts, rat fibroblasts, porcine fibroblasts, etc.,can be used. In some embodiments, the fibroblasts are human fibroblasts.In other embodiments, the fibroblasts are mouse fibroblasts. In otherembodiments, the fibroblasts are rat fibroblasts. Thus, a “post-natalfibroblast” refers to a fibroblast obtained from a post-natal mammal, orthe progeny of a fibroblast obtained from a post-natal mammal.

The post-natal fibroblasts can be from any of a variety of tissuesources. For example, cardiac fibroblasts, foreskin fibroblasts, dermalfibroblasts, lung fibroblasts, etc.

The fibroblasts can be obtained from a living individual. Thefibroblasts can be obtained from tissue taken from a living individual.The fibroblasts can be obtained from a recently deceased individual whois considered a suitable organ donor. In some embodiments, theindividual is screened for various genetic disorders, viral infections,etc., to determine whether the individual is a suitable source offibroblasts, where individuals may be excluded on the basis of one ormore of a genetic disorder, a viral infection, etc.

Suitable fibroblasts express markers characteristic of fibroblasts,where such markers include, e.g., vimentin, prolyl-4-hydroxylase (anintracellular enzyme involved in collagen synthesis),fibroblast-specific protein-1 (see, e.g., Strutz et al. (1995) J. CellBiol. 130:393), fibroblast surface antigen, and collagen type 1. In someembodiments, the fibroblasts used as host cells are cardiac fibroblasts,where cardiac fibroblasts can be characterized as Thy1⁺, vimentin⁺, andare also negative for c-kit or equivalent of c-kit.

In general, a fibroblast that is suitable for use as a host cell formodification or genetic modification in accordance with a subject methodis non-transformed (e.g., exhibits normal cell proliferation), and isotherwise normal.

Where the host cells for modification or genetic modification is apopulation of fibroblasts, the population of fibroblasts are isolated,e.g., the population of fibroblasts is composed of at least about 75%fibroblasts, at least about 80% fibroblasts, at least about 85%fibroblasts, at least about 90% fibroblasts, at least about 95%fibroblasts, at least about 98% fibroblasts, at least about 99%fibroblasts, or greater than 99% fibroblasts.

Post-natal fibroblasts can be derived from tissue of a non-embryonicsubject, a neonatal infant, a child, or an adult. Post-natal fibroblastscan be derived from neonatal or post-natal tissue collected from asubject within the period from birth, including cesarean birth, todeath. For example, the post-natal fibroblasts used to generate inducedcardiomyocytes can be from a subject who is greater than about 10minutes old, greater than about 1 hour old, greater than about 1 dayold, greater than about 1 month old, greater than about 2 months old,greater than about 6 months old, greater than about 1 year old, greaterthan about 2 years old, greater than about 5 years old, greater thanabout 10 years old, greater than about 15 years old, greater than about18 years old, greater than about 25 years old, greater than about 35years old, >45 years old, >55 years old, >65 years old, >80 years old,<80 years old, <70 years old, <60 years old, <50 years old, <40 yearsold, <30 years old, <20 years old or <10 years old.

Methods of isolating fibroblasts from tissues are known in the art, andany known method can be used. As a non-limiting example, cardiacfibroblasts can be obtained using the method of Ieda et al. (2009) Dev.Cell 16:233, or as described in Example 1. Foreskin fibroblasts can beobtained from foreskin tissue (i.e., the skin tissue covering the glanspenis; preputium penis) of a male individual, e.g., from an 8-14 day oldmale individual. The fibroblasts can be obtained by mincing the foreskintissue, then dissociating the tissue to single cells. Foreskin cellclamps can be dissociated by any means known in the art includingphysical de-clamping or enzymatic digestion using, for example trypsin.

As noted above, a post-natal fibroblast is genetically modified with oneor more nucleic acids comprising nucleotide sequences encoding one ormore reprogramming factors (e.g., one or more of Gata4, Mef2c, Tbx5,Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides),or is modified by introducing one or more reprogramming factorpolypeptides themselves (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1,Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset of reprogrammingfactors comprising Gata4, Mef2c, and Tbx5 polypeptides) into thepost-natal fibroblast. Amino acid sequences of such reprogrammingfactors are known in the art. Nucleotide sequences encodingreprogramming factors are known in the art.

Reprogramming Factors

As discussed above, a post-natal fibroblast is genetically modified withone or more nucleic acids comprising nucleotide sequences encoding oneor more reprogramming factors (e.g., one or more of Gata4, Mef2c, Tbx5,Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides),or a post-natal fibroblast is modified by introducing one or morereprogramming factor polypeptides themselves (e.g., one or more ofGata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or asubset of reprogramming factors comprising Gata4, Mef2c, and Tbx5polypeptides) into the post-natal fibroblast.

In some embodiments, the one or more reprogramming factors includes 1,2, 3, 4, 5, 6, 7, 8, or 9 of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1,Myocd, Smyd1, and Srf. In some embodiments, the one or morereprogramming factors includes all of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf. In some embodiments, the one or morereprogramming factors a subset of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf. Exemplary subsets include:

1) Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf;

2) Gata4, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf;

3) Gata4, Mef2c, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf;

4) Gata4, Mef2c, Tbx5, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf;

5) Gata4, Mef2c, Tbx5, Mesp1, Isl-1, Myocd, Smyd1, and Srf;

6) Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Myocd, Smyd1, and Srf;

7) Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Smyd1, and Srf;

8) Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, and Srf;

9) Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, and Smyd1;

10) Gata4, Mef2c, Mesp1, Myocd, Nkx2-5, and Tbx5;

11) Mef2c, Mesp1, Myocd, Nkx2-5, and Tbx5;

12) Gata4, Mesp1, Myocd, Nkx2-5, and Tbx5;

13) Gata4, Mef2c, Myocd, Nkx2-5, and Tbx5;

14) Gata4, Mef2c, Mesp1, Nkx2-5, and Tbx5;

15) Gata4, Mef2c, Mesp1, Myocd, and Tbx5;

16) Gata4, Mef2c, Mesp1, Myocd, and Nkx2-5;

17) Mef2c, Mesp1, Myocd, and Tbx5;

18) Gata4, Mesp1, Myocd, and Tbx5;

19) Gata4, Mef2c, Myocd, and Tbx5;

20) Gata4, Mef2c, Mesp1, and Tbx5;

21) Gata4, Mef2c, Mesp1, and Myocd;

22) Mef2c, Mesp1, and Tbx5;

23) Gata4, Mef2c, and Tbx5.

As indicated above, in some embodiments, the subset of reprogrammingfactors is Gata4, Mef2c, and Tbx5.

Gata4

A Gata4 polypeptide is a member of the GATA family zinc-fingertranscription factor that recognizes and binds a GATA motif (e.g.,recognizes and binds the consensus sequence 5′-AGATAG-3′) present in thepromoter region of many genes. See, e.g., Huang et al. (1995) Gene155:219. Amino acid sequences for Gata4 polypeptides, and nucleotidesequences encoding Gata4 polypeptides, from a variety of species areknown in the art. See, e.g.: 1) GenBank Accession No. NP_002043.2 (Homosapiens Gata4 amino acid sequence; and GenBank Accession No. NM_002052(Homo sapiens Gata4-encoding nucleotide sequence; 2) GenBank AccessionNo. NP_0032118 (Mus musculus Gata4 amino acid sequence); and GenBankAccession No. NM_008092 (Mus musculus Gata4-encoding nucleotidesequence); 3) GenBank Accession No. NP_653331 (Rattus norvegicus Gata4amino acid sequence); and GenBank Accession No. NM_144730 (Rattusnorvegicus Gata4-encoding nucleotide sequence); 4) GenBank Accession No.ABI63575 (Danio rerio Gata4 amino acid sequence; and GenBank AccessionNo. DQ886664 (Danio rerio Gata4-encoding nucleotide sequence; and 5)GenBank Accession No. AAH71101.1 (Xenopus laevis Gata4 amino acidsequence); and GenBank Accession No. BC071107 (Xenopus laevisGata4-encoding nucleotide sequence).

In some embodiments, a suitable Gata4 nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, nucleotide sequence identity to a contiguous stretch offrom about 900 nucleotides to about 1000 nucleotides (nt), from about1000 nt to about 1100 nt, from about 1100 nt to about 1200 nt, or fromabout 1200 nt to 1329 nt, of the nucleotide sequence depicted in SEQ IDNO:14. In some embodiments, a suitable Gata4 nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, nucleotide sequence identity to a contiguous stretch offrom about 900 nucleotides to about 1000 nucleotides (nt), from about1000 nt to about 1100 nt, from about 1100 nt to about 1200 nt, or fromabout 1200 nt to 1323 nt, of the nucleotide sequence depicted in SEQ IDNO:28.

A suitable Gata4 nucleic acid comprises a nucleotide sequence encoding aGata4 polypeptide, where in some embodiments, a suitable Gata4polypeptide comprises an amino acid sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toa contiguous stretch of from about 350 amino acids (aa) to about 400 aa,or from about 400 aa to 442 aa, of the amino acid sequence depicted inSEQ ID NO:13. In some embodiments, a suitable Gata4 polypeptidecomprises an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to acontiguous stretch of from about 350 amino acids (aa) to about 400 aa,or from about 400 aa to 441 aa, of the amino acid sequence depicted inSEQ ID NO:27. The encoded Gata4 polypeptide is biologically active,e.g., recognizes and binds a GATA motif (e.g., recognizes and binds theconsensus sequence 5′-AGATAG-3′) present in a promoter; and activatestranscription of a gene operably linked to the promoter comprising theGATA motif.

In some embodiments, a polypeptide that is functionally equivalent to aGata4 polypeptide (or a nucleotide sequence encoding such functionalequivalent) is used. For example, in some embodiments, a Gata5polypeptide (or a nucleotide sequence encoding a Gata5 polypeptide) isused. In other embodiments, a Gata6 polypeptide (or a nucleotidesequence encoding a Gata6 polypeptide) is used.

Amino acid sequences of Gata5 polypeptides, and nucleotide sequencesencoding Gata5 polypeptides, are known in the art. See, e.g., GenBankAccession Nos.: 1) NP_536721 (Homo sapiens Gata5 amino acid sequence),and NM_080473 (nucleotide sequence encoding the NP_536721 amino acidsequence); 2) NP_032119 (Mus musculus Gata5 amino acid sequence), andNM_008093 (nucleotide sequence encoding the NP_032119 amino acidsequence); and 3) NP_001019487 (Rattus norvegicus Gata5 amino acidsequence), and NM_001024316 (nucleotide sequence encoding theNP_001019487 amino acid sequence).

Amino acid sequences of Gata6 polypeptides, and nucleotide sequencesencoding Gata6 polypeptides, are known in the art. See, e.g., GenBankAccession Nos.: 1) NP_005248 (Homo sapiens Gata6 amino acid sequence),and NM_005257 (nucleotide sequence encoding the NP_005248 amino acidsequence); 2) NP_062058 (Rattus norvegicus Gata6 amino acid sequence)and NM_019185 (nucleotide sequence encoding the NP_062058 amino acidsequence); 3) NP_034388 (Mus musculus Gata6 amino acid sequence), andNM_010258 (nucleotide sequence encoding the NP_034388 amino acidsequence).

In some embodiments, a suitable functional equivalent of a Gata4polypeptide is a polypeptide having at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to an amino acidsequence of a Gata5 polypeptide or a Gata6 polypeptide.

In some embodiments, a suitable nucleotide sequence encoding afunctional equivalent of a Gata4 polypeptide comprises a nucleotidesequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, nucleotide sequence identity to a nucleotide sequence encoding aGata5 polypeptide or a Gata6 polypeptide.

Mef2c

Mef2c (myocyte-specific enhancer factor 2c) is a transcription activatorthat binds specifically to the MEF2 element (e.g., the consensussequence: 5′-CT(A/t)(a/t)AAATAG-3′) (SEQ ID NO:10) present in theregulatory regions of many muscle-specific genes. See, e.g., Andrés etal. (1995) J. Biol. Chem. 270:23246. Mef2c can include one or morepost-translational modifications, e.g., phosphorylation on Ser-59 andSer-396; sumoylation on Lys-391; and acetylation on Lys-4.

Amino acid sequences of Mef2c polypeptides, and nucleotide sequencesencoding Mef2c polypeptides, from a variety of species are known in theart. See, e.g.: 1) GenBank Accession No. XP_001056692 (Rattus norvegicusMef2c amino acid sequence); and GenBank Accession No. XM_001056692(Rattus norvegicus Mef2c-encoding nucleotide sequence); 2) GenBankAccession No. NP_079558.1 (Mus musculus Mef2c isoform 2 amino acidsequence); and GenBank Accession No. NM_025282 (Mus musculus Mef2cisoform 2-encoding nucleotide sequence); 3) GenBank Accession No.NP_001164008 (Mus musculus Mef2c isoform 1 amino acid sequence); andGenBank Accession No. NM_001170537 (Mus musculus Mef2c isoform1-encoding nucleotide sequence); 4) GenBank Accession No. NP_001124477(Homo sapiens Mef2c isoform 2 amino acid sequence); and GenBankAccession No. NM_001131005 (Homo sapiens Mef2c isoform 2-encodingnucleotide sequence); 5) GenBank Accession No. NP_002388 (Homo sapiensMef2c isoform 1 amino acid sequence); and GenBank Accession No.NM_002397 (Homo sapiens Mef2c isoform 1-encoding nucleotide sequence).

In some embodiments, a suitable Mef2c nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, nucleotide sequence identity to a contiguous stretch offrom about 900 nucleotides to about 1000 nucleotides (nt), from about1000 nt to about 1100 nt, from about 1100 nt to about 1200 nt, fromabout 1200 nt to 1300 nt, or from about 1300 nt to 1392 nt, of thenucleotide sequence depicted in SEQ ID NO:16.

In some embodiments, a suitable Mef2c nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, nucleotide sequence identity to a contiguous stretch offrom about 900 nucleotides to about 1000 nucleotides (nt), from about1000 nt to about 1100 nt, from about 1100 nt to about 1200 nt, fromabout 1200 nt to 1300 nt, or from about 1300 nt to 1422 nt, of thenucleotide sequence depicted in SEQ ID NO:18. In some embodiments, asuitable Mef2c nucleic acid comprises a nucleotide sequence having atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, nucleotidesequence identity to a contiguous stretch of from about 900 nucleotidesto about 1000 nucleotides (nt), from about 1000 nt to about 1100 nt,from about 1100 nt to about 1200 nt, or from about 1200 nt to 1296 nt,of the nucleotide sequence depicted in SEQ ID NO:23.

A suitable Mef2c nucleic acid comprises a nucleotide sequence encoding aMef2c polypeptide, where in some embodiments a suitable Mef2cpolypeptide comprises an amino acid sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toa contiguous stretch of from about 350 amino acids (aa) to about 400 aa,or from about 400 aa to 463 aa, of the amino acid sequence depicted inSEQ ID NO:15. The encoded Mef2c polypeptide is biologically active,e.g., recognizes and binds a MEF2C element in a promoter; and activatestranscription of a gene operably linked to the promoter.

In some embodiments, a suitable Mef2c polypeptide comprises an aminoacid sequence having at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or 100%, amino acid sequence identity to a contiguous stretch of fromabout 350 amino acids (aa) to about 400 aa, or from about 400 aa to 432aa, of the amino acid sequence depicted in SEQ ID NO:24. The encodedMef2c polypeptide is biologically active, e.g., recognizes and binds aMEF2C element in a promoter; and activates transcription of a geneoperably linked to the promoter.

A suitable Mef2c nucleic acid comprises a nucleotide sequence encoding aMef2c polypeptide, where a suitable Mef2c polypeptide comprises an aminoacid sequence having at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or 100%, amino acid sequence identity to a contiguous stretch of fromabout 350 amino acids (aa) to about 400 aa, or from about 400 aa to 473aa, of the amino acid sequence depicted in SEQ ID NO:17. The encodedMef2c polypeptide is biologically active, e.g., recognizes and binds aMEF2C element in a promoter; and activates transcription of a geneoperably linked to the promoter.

In some embodiments, a polypeptide that is functionally equivalent to aMef2c polypeptide (or a nucleotide sequence encoding such functionalequivalent) is used. For example, in some embodiments, a Mef2apolypeptide (or a nucleotide sequence encoding a Mef2a polypeptide) isused. In other embodiments, a Mef2b polypeptide (or a nucleotidesequence encoding a Mef2b polypeptide) is used. In other embodiments, aMef2d polypeptide (or a nucleotide sequence encoding a Mef2dpolypeptide) is used.

Amino acid sequences of Mef2a, Me2b, and Mef2d polypeptides are known,as are nucleotide sequences encoding Mef2a, Me2b, and Mef2dpolypeptides. See, e.g., GenBank Accession Nos.: 1) NP_005578.2 (Homosapiens Mef2a isoform 1 amino acid sequence), and NM_005587 (nucleotidesequence encoding the NP_005578.2 amino acid sequence); 2)NP_001124398.1 (Homo sapiens Mef2a isoform 2 amino acid sequence), andNM_001130926 (nucleotide sequence encoding the NP_001124398.1 amino acidsequence); 3) NP_001124399.1 (Homo sapiens Mef2a isoform 3 amino acidsequence), and NM_001130927 (nucleotide sequence encoding theNP_001124399.1 amino acid sequence); 4) NP_001124400.1 (Homo sapiensMef2a isoform 4 amino acid sequence), and NM_001130928 (nucleotidesequence encoding the NP_001124400.1 amino acid sequence); 5)NP_001139257.1 (Homo sapiens Mef2b isoform a amino acid sequence), andNM_001145785 (nucleotide sequence encoding the NP_001139257.1 amino acidsequence); 6) NP_005910.1 (Homo sapiens Mef2b isoform b amino acidsequence), and NM_005919 (nucleotide sequence encoding the NP_005910.1amino acid sequence); 7) NP_032604.2 (Mus musculus Mef2b isoform 1 aminoacid sequence), and NM_008578 (nucleotide sequence encoding theNP_032604.2 amino acid sequence); 8) NP_001038949.1 (Mus musculus Mef2bisoform 2 amino acid sequence), and NM_001045484 (nucleotide sequenceencoding the NP_001038949.1 amino acid sequence); 9) NP_005911.1 (Homosapiens Mef2d amino acid sequence), and NM_005920 (nucleotide sequenceencoding the NP_005911.1 amino acid sequence); and 10) NP_598426.1 (Musmusculus Mef2d amino acid sequence), and NM_133665 (nucleotide sequenceencoding the NP_598426.1 amino acid sequence).

In some embodiments, a suitable functional equivalent of a Mef2cpolypeptide is a polypeptide having at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to an amino acidsequence of a Mef2a polypeptide, a Mef2b polypeptide, or a Mef2dpolypeptide.

In some embodiments, a suitable nucleotide sequence encoding afunctional equivalent of a Mef2c polypeptide comprises a nucleotidesequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, nucleotide sequence identity to a nucleotide sequence encoding aMef2a polypeptide, a Mef2b polypeptide, or a Mef2d polypeptide.

Tbx5

Tbx5 (T-box transcription factor 5) is a transcription factor that bindsto an recognizes a T-box (e.g., an element having the consensus sequence5′-(A/G)GGTGT-3′) in the promoter region of some genes; and activatestranscription of genes operably linked to such promoters.

Amino acid sequences for Tbx5 polypeptides, and nucleotide sequencesencoding Tbx5 polypeptides, from a variety of species are known in theart. See, e.g.: 1) GenBank Accession No. CAA70592.1 (Homo sapiens Tbx5amino acid sequence); and GenBank Accession No. Y09445 (Homo sapiensTbx5-encoding nucleotide sequence); 2) GenBank Accession No. NP_000183(Homo sapiens Tbx5 amino acid sequence); and GenBank Accession No.NM_000192 (Homo sapiens Tbx5-encoding nucleotide sequence); 3) GenBankAccession No. NP_001009964.1 (Rattus norvegicus Tbx5 amino acidsequence); and GenBank Accession No. NM_001009964 (Rattus norvegicusTbx5-encoding nucleotide sequence; 4) GenBank Accession No. NP_035667(Mus musculus Tbx5 amino acid sequence); and NM_011537 (Mus musculusTbx5-encoding nucleotide sequence); 5) GenBank Accession No.NP_001079170 (Xenopus laevis Tbx5 amino acid sequence); and GenBankAccession No. NM_001085701 (Xenopus laevis Tbx5-encoding nucleotidesequence).

In some embodiments, a suitable Tbx5 nucleic acid comprises a nucleotidesequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, nucleotide sequence identity to a contiguous stretch of from about900 nucleotides to about 1000 nucleotides (nt), from about 1200 nt to1300 nt, from about 1300 nt to about 1400 nt, or from about 1400 nt toabout 1500 nt, or from about 1500 nt to 1542 nt of the nucleotidesequence depicted in SEQ ID NO:20. In some embodiments, a suitable Tbx5nucleic acid comprises a nucleotide sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, nucleotide sequence identity toa contiguous stretch of from about 900 nucleotides to about 1000nucleotides (nt), from about 1200 nt to 1300 nt, from about 1300 nt toabout 1400 nt, or from about 1400 nt to about 1500 nt, or from about1500 nt to 1560 nt of the nucleotide sequence depicted in SEQ ID NO:25.

In some embodiments, a suitable Tbx5 nucleic acid comprises a nucleotidesequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, nucleotide sequence identity to a contiguous stretch of from about900 nucleotides to about 1000 nucleotides (nt), from about 1200 nt to1300 nt, from about 1300 nt to about 1400 nt, or from about 1400 nt toabout 1500 nt, or from about 1500 nt to 1557, of the nucleotide sequencedepicted in SEQ ID NO:22.

A suitable Tbx5 nucleic acid comprises a nucleotide sequence encoding aTbx5 polypeptide. In some embodiments, a suitable Tbx5 polypeptidecomprises an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to acontiguous stretch of from about 350 amino acids (aa) to about 400 aa,from about 400 aa to about 500 aa, or from about 500 aa to 513 aa, ofthe amino acid sequence depicted in SEQ ID NO:19. In some embodiments, asuitable Tbx5 polypeptide comprises an amino acid sequence having atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to a contiguous stretch of from about 350 amino acids(aa) to about 400 aa, from about 400 aa to about 500 aa, or from about500 aa to 518 aa, of the amino acid sequence depicted in SEQ ID NO:26.The encoded Tbx5 polypeptide is biologically active, e.g., recognizesand binds a Tbx5 binding site (e.g., an element having the consensussequence 5′-(A/G)GGTGT-3′) in a promoter; and activates transcription ofa gene operably linked to the promoter.

A suitable Tbx5 nucleic acid comprises a nucleotide sequence encoding aTbx5 polypeptide, where in some embodiments a suitable Tbx5 polypeptidecomprises an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to acontiguous stretch of from about 350 amino acids (aa) to about 400 aa,from about 400 aa to about 500 aa, or from about 500 aa to 518 aa, ofthe amino acid sequence depicted in SEQ ID NO:21. The encoded Tbx5polypeptide is biologically active, e.g., recognizes and binds a Tbx5binding site (e.g., an element having the consensus sequence5′-(A/G)GGTGT-3′) in a promoter; and activates transcription of a geneoperably linked to the promoter.

Mesp1

In some embodiments, a suitable mesoderm posterior protein 1 (Mesp1)nucleic acid comprises a nucleotide sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, nucleotide sequence identity toa contiguous stretch of from about 600 nucleotides to about 800nucleotides (nt), or 804 nt, of the nucleotide sequence depicted in SEQID NO:30.

A suitable Mesp1 nucleic acid comprises a nucleotide sequence encoding aMesp1 polypeptide, where in some embodiments, a suitable Mesp1polypeptide comprises an amino acid sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toa contiguous stretch of from about 200 amino acids (aa) to about 250 aa,or from about 250 aa to 268 aa, of the amino acid sequence depicted inSEQ ID NO:29. The encoded Mesp1 polypeptide is biologically active.

Nkx2-5

In some embodiments, a suitable Nkx2-5 nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, nucleotide sequence identity to a contiguous stretch offrom about 350 nucleotides to about 450 nucleotides (nt), or 456 nt, ofthe nucleotide sequence depicted in SEQ ID NO:32. In some embodiments, asuitable Nkx2-5 nucleic acid comprises a nucleotide sequence having atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, nucleotidesequence identity to a contiguous stretch of from about 850 nucleotidesto about 950 nucleotides (nt), or 975 nt, of the nucleotide sequencedepicted in SEQ ID NO:34. In some embodiments, a suitable Nkx2-5 nucleicacid comprises a nucleotide sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, nucleotide sequence identity to acontiguous stretch of from about 230 nucleotides to about 330nucleotides (nt), or 339 nt, of the nucleotide sequence depicted in SEQID NO:36.

A suitable Nkx2-5 nucleic acid comprises a nucleotide sequence encodinga Nkx2-5 polypeptide, where in some embodiments, a suitable Nkx2-5polypeptide comprises an amino acid sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toa contiguous stretch of from about 125 amino acids (aa) to about 150 aa,of the amino acid sequence depicted in SEQ ID NO:31. In someembodiments, a suitable Nkx2-5 polypeptide comprises an amino acidsequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from about275 amino acids (aa) to about 300 aa, or from about 300 aa to about 324aa, of the amino acid sequence depicted in SEQ ID NO:33. In someembodiments, a suitable Nkx2-5 polypeptide comprises an amino acidsequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from about75 amino acids (aa) to about 100 aa, or from about 100 aa to about 112aa, of the amino acid sequence depicted in SEQ ID NO:35. The encodedNkx2-5 polypeptide is biologically active.

Isl-1

In some embodiments, a suitable islet-1 (Isl-1) nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, nucleotide sequence identity to a contiguous stretch offrom about 900 nucleotides to about 1000 nucleotides (nt), or 1050 nt,of the nucleotide sequence depicted in SEQ ID NO:38.

A suitable Isl-1 nucleic acid comprises a nucleotide sequence encoding aIsl-1 polypeptide, where in some embodiments, a suitable Isl-1polypeptide comprises an amino acid sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toa contiguous stretch of from about 300 amino acids (aa) to about 325 aa,or from about 325 aa to 346 aa, of the amino acid sequence depicted inSEQ ID NO:37. The encoded Isl-1 polypeptide is biologically active.

Myocd

In some embodiments, a suitable myocardin (Myocd) nucleic acid comprisesa nucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, nucleotide sequence identity to a contiguous stretch offrom about 1500 nucleotides to about 2000 nucleotides (nt), or 2055 nt,of the nucleotide sequence depicted in SEQ ID NO:40. In someembodiments, a suitable Myocd nucleic acid comprises a nucleotidesequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, nucleotide sequence identity to a contiguous stretch of from about2500 nucleotides to about 2900 nucleotides (nt), or 2961 nt, of thenucleotide sequence depicted in SEQ ID NO:42. In some embodiments, asuitable Myocd nucleic acid comprises a nucleotide sequence having atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, nucleotidesequence identity to a contiguous stretch of from about 2500 nucleotidesto about 2800 nucleotides (nt), or 2871 nt, of the nucleotide sequencedepicted in SEQ ID NO:44.

A suitable Myocd nucleic acid comprises a nucleotide sequence encoding aMyocd polypeptide, where in some embodiments, a suitable Myocdpolypeptide comprises an amino acid sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toa contiguous stretch of from about 600 amino acids (aa) to about 650 aa,or from about 650 aa to 684 aa, of the amino acid sequence depicted inSEQ ID NO:39. In some embodiments, a suitable Myocd polypeptidecomprises an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to acontiguous stretch of from about 900 amino acids (aa) to about 950 aa,or from about 950 aa to 986 aa, of the amino acid sequence depicted inSEQ ID NO:41. In some embodiments, a suitable Myocd polypeptidecomprises an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to acontiguous stretch of from about 900 amino acids (aa) to about 925 aa,or from about 925 aa to 938 aa, of the amino acid sequence depicted inSEQ ID NO:43. The encoded Myocd polypeptide is biologically active.

Smyd1

In some embodiments, a suitable Smyd1 nucleic acid comprises anucleotide sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, nucleotide sequence identity to a contiguous stretch offrom about 1400 nucleotides to about 1450 nucleotides (nt), or fromabout 1450 nt to 1473 nt, of the nucleotide sequence depicted in SEQ IDNO:46.

A suitable Smyd1 nucleic acid comprises a nucleotide sequence encoding aSmyd1 polypeptide, where in some embodiments, a suitable Smyd1polypeptide comprises an amino acid sequence having at least about 80%,at least about 85%, at least about 90%, at least about 95%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identity toa contiguous stretch of from about 400 amino acids (aa) to about 450 aa,or from about 450 aa to 490 aa, of the amino acid sequence depicted inSEQ ID NO:45. The encoded Smyd1 polypeptide is biologically active.

Srf

In some embodiments, a suitable Srf nucleic acid comprises a nucleotidesequence having at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, nucleotide sequence identity to a contiguous stretch of from about1450 nucleotides to about 1500 nucleotides (nt), or from about 1500 ntto 1527 nt, of the nucleotide sequence depicted in SEQ ID NO:48.

A suitable Srf nucleic acid comprises a nucleotide sequence encoding aSrf polypeptide, where in some embodiments, a suitable Srf polypeptidecomprises an amino acid sequence having at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to acontiguous stretch of from about 450 amino acids (aa) to about 500 aa,or from about 500 aa to 508 aa, of the amino acid sequence depicted inSEQ ID NO:47. The encoded Srf polypeptide is biologically active.

It has been found that introduction of one or more nucleic acidscomprising nucleotide sequences encoding one or more reprogrammingfactors (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1,Myocd, Smyd1, and Srf; or a subset of reprogramming factors comprisingGata4, Mef2c, and Tbx5 polypeptides) is sufficient to reprogram apost-natal fibroblast into a cardiomyocyte. Thus, a post-natalfibroblast can be reprogrammed to become a cardiomyocyte without theneed for introducing an induction factor (e.g., any other exogenouspolypeptide; any other nucleic acid encoding any other exogenouspolypeptide) that would reprogram a fibroblast into a stem cell orprogenitor cell into the post-natal fibroblast. For example, a subjectmethod does not require and does not involve introducing into apost-natal fibroblast any of an exogenous Sox2 polypeptide, an exogenousOct-3/4 polypeptide, an exogenous c-Myc polypeptide, an exogenous Klf4polypeptide, an exogenous Nanog polypeptide, or an exogenous Lin28polypeptide. A subject method does not require and does not involveintroducing into a post-natal fibroblast a nucleic acid(s) comprisingnucleotide sequences encoding any of Sox2, Oct-3/4, c-Myc, Klf4, Nanog,or any other polypeptide that would reprogram a fibroblast into a stemcell or progenitor cell.

As noted above, to generate an induced cardiomyocyte, a post-natalfibroblast is genetically modified with one or more nucleic acidscomprising nucleotide sequences encoding one or more reprogrammingfactors (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1,Myocd, Smyd1, and Srf; or a subset of reprogramming factors comprisingGata4, Mef2c, and Tbx5 polypeptides). Induced cardiomyocytes express oneor more cardiomyocyte-specific markers, where cardiomyocyte-specificmarkers include, but are not limited to, cardiac troponin I, cardiactroponin-C, tropomyosin, caveolin-3, myosin heavy chain, myosin lightchain-2a, myosin light chain-2v, ryanodine receptor, sarcomericα-actinin, Nkx2.5, connexin 43, and atrial natriuretic factor. Inducedcardiomyocytes can also exhibit sarcomeric structures. Inducedcardiomyocytes exhibit increased expression of cardiomyocyte-specificgenes Actc1 (cardiac α-actin), Myh6 (α-myosin heavy chain), Ryr2(ryanodine receptor 2), and Gja1 (connexin43). Expression of fibroblastsmarkers such as Colla2 (collagen 1a2) is downregulated in inducedcardiomyocytes, compared to fibroblasts from which the iCM is derived.

The expression of various markers specific to cardiomyocytes is detectedby conventional biochemical or immunochemical methods (e.g.,enzyme-linked immunosorbent assay; immunohistochemical assay; and thelike). Alternatively, expression of nucleic acid encoding acardiomyocyte-specific marker can be assessed. Expression ofcardiomyocyte-specific marker-encoding nucleic acids in a cell can beconfirmed by reverse transcriptase polymerase chain reaction (RT-PCR) orhybridization analysis, molecular biological methods which have beencommonly used in the past for amplifying, detecting and analyzing mRNAcoding for any marker proteins. Nucleic acid sequences coding formarkers specific to cardiomyocytes are known and are available throughpublic data bases such as GenBank; thus, marker-specific sequencesneeded for use as primers or probes is easily determined.

Induced cardiomyocytes can also exhibit spontaneous contraction. Whetheran induced cardiomyocyte exhibits spontaneous contraction can bedetermined using standard electrophysiological methods (e.g., patchclamp); a suitable method is described in the Examples.

Induced cardiomyocytes can also exhibit spontaneous Ca²⁺ oscillations.Ca²⁺ oscillations can be detected using standard methods, e.g., usingany of a variety of calcium-sensitive dyes. intracellular Ca²⁺ion-detecting dyes include, but are not limited to, fura-2, bis-fura 2,indo-1, Quin-2, Quin-2 AM, Benzothiaza-1, Benzothiaza-2, indo-5F,Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1, fluo-3, rhod-2,rhod-3, fura-4F, fura-5F, fura-6F, fluo-4, fluo-5F, fluo-5N, OregonGreen 488 BAPTA, Calcium Green, Calcein, Fura-C18, Calcium Green-C18,Calcium Orange, Calcium Crimson, Calcium Green-5N, Magnesium Green,Oregon Green 488 BAPTA-1, Oregon Green 488 BAPTA-2, X-rhod-1, Fura Red,Rhod-5F, Rhod-5N, X-Rhod-5N, Mag-Rhod-2, Mag-X-Rhod-1, Fluo-5N, Fluo-5F,Fluo-4FF, Mag-Fluo-4, Aequorin, dextran conjugates or any otherderivatives of any of these dyes, and others (see, e.g., the catalog orInternet site for Molecular Probes, Eugene, see, also, Nuccitelli, ed.,Methods in Cell Biology, Volume 40: A Practical Guide to the Study ofCalcium in Living Cells, Academic Press (1994); Lambert, ed., CalciumSignaling Protocols (Methods in Molecular Biology Volume 114), HumanaPress (1999); W. T. Mason, ed., Fluorescent and Luminescent Probes forBiological Activity. A Practical Guide to Technology for QuantitativeReal-Time Analysis, Second Ed, Academic Press (1999); Calcium SignalingProtocols (Methods in Molecular Biology), 2005, D. G. Lamber, ed.,Humana Press).

Introduction of Exogenous Re-Programming Factor Polypeptide into aPost-Natal Fibroblast

In some embodiments, introduction of exogenous reprogramming factorpolypeptides (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf; or a subset of reprogramming factorscomprising Gata4, Mef2c, and Tbx5 polypeptides) into a post-natalfibroblast is achieved by contacting the post-natal fibroblast withexogenous reprogramming factor polypeptides, wherein the exogenousreprogramming factor polypeptides are taken up into the cell.

In some embodiments, each of an exogenous reprogramming factorpolypeptides comprises a protein transduction domain. As a non-limitingexample, an exogenous Gata4 polypeptide, an exogenous Mef2C polypeptide,and a Tbx4 polypeptide is linked, covalently or non-covalently, to aprotein transduction domain.

“Protein Transduction Domain” or PTD refers to a polypeptide,polynucleotide, carbohydrate, or organic or inorganic compound thatfacilitates traversing a lipid bilayer, micelle, cell membrane,organelle membrane, or vesicle membrane. A PTD attached to anothermolecule facilitates the molecule traversing a membrane, for examplegoing from extracellular space to intracellular space, or cytosol towithin an organelle. In some embodiments, a PTD is covalently linked tothe amino terminus of a reprogramming factor polypeptide (e.g., a Gata4polypeptide, a Mef2c polypeptide, or a Tbx5 polypeptide). In someembodiments, a PTD is covalently linked to the carboxyl terminus of areprogramming factor polypeptide (e.g., a Gata4 polypeptide, a Mef2cpolypeptide, or a Tbx5 polypeptide).

Exemplary protein transduction domains include but are not limited to aminimal undecapeptide protein transduction domain (corresponding toresidues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO:1); apolyarginine sequence comprising a number of arginines sufficient todirect entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50arginines); a VP22 domain (Zender et al., Cancer Gene Ther. 2002 June;9(6):489-96); an Drosophila Antennapedia protein transduction domain(Noguchi et al., Diabetes 2003; 52(7):1732-1737); a truncated humancalcitonin peptide (Trehin et al. Pharm. Research, 21:1248-1256, 2004);polylysine (Wender et al., PNAS, Vol. 97:13003-13008); RRQRRTSKLMKR (SEQID NO:2); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:3);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:4); and RQIKIWFQNRRMKWKK(SEQ ID NO:5). Exemplary PTDs include but are not limited to,YGRKKRRQRRR (SEQ ID NO:1), RKKRRQRRR (SEQ ID NO:6); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;Exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following: YGRKKRRQRRR (SEQ ID NO:1); RKKRRQRR (SEQ IDNO:6); YARAAARQARA (SEQ ID NO:7); THRLPRRRRRR (SEQ ID NO:8); andGGRRARRRRRR (SEQ ID NO:9).

In some embodiments, an exogenous reprogramming factor polypeptide(e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd,Smyd1, and Srf; or a subset of reprogramming factors comprising Gata4,Mef2c, and Tbx5 polypeptides) comprises an arginine homopolymer of from3 arginine residues to 50 arginine residues, e.g., from 3 to 6 arginineresidues, from 6 to 10 arginine residues, from 10 to 20 arginineresidues, from 20 to 30 arginine residues, from 30 to 40 arginineresidues, or from 40 to 50 arginine residues. In some embodiments, anexogenous reprogramming factor polypeptide (e.g., one or more of Gata4,Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides)comprises six Arg residues covalently linked (e.g., by a peptide bond)at the amino terminus of the reprogramming factor polypeptide. In someembodiments, an exogenous reprogramming factor polypeptide comprises sixArg residues covalently linked (e.g., by a peptide bond) at the carboxylterminus of the reprogramming factor polypeptide.

Exogenous reprogramming factor polypeptides (e.g., one or more of Gata4,Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf; or a subset ofreprogramming factors comprising Gata4, Mef2c, and Tbx5 polypeptides)that are introduced into a host post-natal fibroblast can be purified,e.g., at least about 75% pure, at least about 80% pure, at least about85% pure, at least about 90% pure, at least about 95% pure, at leastabout 98% pure, at least about 99% pure, or more than 99% pure, e.g.,free of proteins other than reprogramming factor(s) being introducedinto the cell and free of macromolecules other than the reprogrammingfactor(s) being introduced into the cell.

Genetic Modification of a Post-Natal Fibroblast

In some embodiments, introduction of exogenous reprogramming factorpolypeptides (e.g., one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf; or a subset of reprogramming factorscomprising Gata4, Mef2c, and Tbx5 polypeptides) into a post-natalfibroblast is achieved by genetic modification of the post-natalfibroblast with one or more exogenous nucleic acids comprisingnucleotide sequences encoding reprogramming factor polypeptides (e.g.,one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1,and Srf; or a subset of reprogramming factors comprising Gata4, Mef2c,and Tbx5 polypeptides). In the following discussion, one or moreexogenous nucleic acids comprising nucleotide sequences encoding one ormore of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf(or a subset of reprogramming factors comprising Gata4, Mef2c, and Tbx5polypeptides) are referred to generically as “one or more exogenousnucleic acids.”

The one or more exogenous nucleic acids comprising nucleotide sequencesencoding the above-noted exogenous reprogramming factor polypeptides canbe a recombinant expression vector, where suitable vectors include,e.g., recombinant retroviruses, lentiviruses, and adenoviruses;retroviral expression vectors, lentiviral expression vectors, nucleicacid expression vectors, and plasmid expression vectors. In some cases,the one or more exogenous nucleic acids is integrated into the genome ofa host post-natal fibroblast and its progeny. In other cases, the one ormore exogenous nucleic acids persists in an episomal state in the hostpost-natal fibroblast and its progeny. In some cases, an endogenous,natural version of the reprogramming factor-encoding nucleic acid mayalready exist in the cell but an additional “exogenous gene” is added tothe host post-natal fibroblast to increase expression of thereprogramming factor. In other cases, the exogenous reprogrammingfactor-encoding nucleic acid encodes a reprogramming factor polypeptidehaving an amino acid sequence that differs by one or more amino acidsfrom a polypeptide encoded by an endogenous reprogrammingfactor-encoding nucleic acid within the host post-natal fibroblast.

In some embodiments, a post-natal fibroblast is genetically modifiedwith three separate expression constructs (expression vectors), eachcomprising a nucleotide sequence encoding one of Gata4, Mef2c, Tbx5,Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf. In some embodiments, anexpression construct will comprise nucleotide sequences encoding two ormore of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf.

In some embodiments, a post-natal fibroblast is genetically modifiedwith three separate expression constructs (expression vectors), eachcomprising a nucleotide sequence encoding one of Gata4, Mef2c, and Tbx5.In some embodiments, an expression construct will comprise nucleotidesequences encoding both Gata4 and Mef2c, both Gata4 and Tbx5, or bothMef2c and Tbx5. In some embodiments, an expression construct willcomprise nucleotide sequences encoding Gata4, Mef2c, and Tbx5.

In some embodiments, one or more exogenous nucleic acids comprisingnucleotide sequences encoding one or more of Gata4, Mef2c, Tbx5, Mesp1,Nkx2-5, Isl-1, Myocd, Smyd1, and Srf (or a subset, e.g., Gata4, Mef2c,and Tbx5) polypeptides is introduced into a single post-natal fibroblast(e.g., a single post-natal fibroblast host cell) in vitro. In otherembodiments, one or more exogenous nucleic acids comprising nucleotidesequences encoding Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd,Smyd1, and Srf (or a subset, e.g., Gata4, Mef2c, and Tbx5) polypeptidesis introduced into a population of post-natal fibroblasts (e.g., apopulation of host post-natal fibroblasts) in vitro. In someembodiments, one or more exogenous nucleic acids comprising nucleotidesequences encoding one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf (or a subset, e.g., Gata4, Mef2c, and Tbx5)polypeptides is introduced into a post-natal fibroblast (e.g., a singlepost-natal fibroblast or a population of post-natal fibroblasts) invivo.

Where a population of post-natal fibroblasts is genetically modified (invitro or in vivo) with one or more exogenous nucleic acids comprisingnucleotide sequences encoding one or more of Gata4, Mef2c, Tbx5, Mesp1,Nkx2-5, Isl-1, Myocd, Smyd1, and Srf (or a subset, e.g., Gata4, Mef2c,and Tbx5) polypeptides, the one or more exogenous nucleic acids can beintroduced into greater than 20% of the total population of post-natalfibroblasts, e.g., 25%, 30%, 35%, 40%, 44%, 50%, 57%, 62%, 70%, 74%,75%, 80%, 90%, or other percent of cells greater than 20%.

In some embodiments, the one or more nucleic acids comprising nucleotidesequences encoding one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf (or a subset, e.g., Gata4, Mef2c, and Tbx5)polypeptides is/are an expression construct that provides for productionof the one or more reprogramming factor polypeptides in the geneticallymodified host post-natal fibroblast cell. In some embodiments, theexpression construct is a viral construct, e.g., a recombinantadeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), arecombinant adenoviral construct, a recombinant lentiviral construct,etc.

Suitable expression vectors include, but are not limited to, viralvectors (e.g. viral vectors based on vaccinia virus; poliovirus;adenovirus (see, e.g., Li et al., Invest Opthalmol V is Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson,PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999;WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther9:8186, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al.,Invest Opthalmol V is Sci 38:2857 2863, 1997; Jomary et al., Gene Ther4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali etal., Hum Mol Genet. 5:591 594, 1996; Srivastava in WO 93/09239, Samulskiet al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988)166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40;herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshiet al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816,1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosisvirus, and vectors derived from retroviruses such as Rous Sarcoma Virus,Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus); and the like.

Numerous suitable expression vectors are known to those of skill in theart, and many are commercially available. The following vectors areprovided by way of example; for eukaryotic host cells: pXT1, pSG5(Stratagene), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, anyother vector may be used so long as it is compatible with the host cell.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation control elements, includingconstitutive and inducible promoters, transcription enhancer elements,transcription terminators, etc. may be used in the expression vector(see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

In some embodiments, a reprogramming factor-encoding nucleotide sequence(e.g., a Gata4-encoding nucleotide sequence, an Mef2c-encodingnucleotide sequence, a Tbx5-encoding nucleotide sequence) is operablylinked to a control element, e.g., a transcriptional control element,such as a promoter. The transcriptional control element is functional ina eukaryotic cell, e.g., a mammalian cell. Suitable transcriptionalcontrol elements include promoters and enhancers. In some embodiments,the promoter is constitutively active. In other embodiments, thepromoter is inducible.

Non-limiting examples of suitable eukaryotic promoters (promotersfunctional in a eukaryotic cell) include CMV immediate early, HSVthymidine kinase, early and late SV40, long terminal repeats (LTRs) fromretrovirus, and mouse metallothionein-I.

In some embodiments, a reprogramming factor-encoding nucleotide sequenceis operably linked to a cardiac-specific transcriptional regulatorelement (TRE), where TREs include promoters and enhancers. Suitable TREsinclude, but are not limited to, TREs derived from the following genes:myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C, andcardiac actin. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbinset al. (1995) Ann. N.Y. Acad. Sci. 752:492-505; Linn et al. (1995) Circ.Res. 76:584-591; Parmacek et al. (1994) Mol. Cell. Biol. 14:1870-1885;Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al.(1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.

Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art. The expression vector may alsocontain a ribosome binding site for translation initiation and atranscription terminator. The expression vector may also includeappropriate sequences for amplifying expression.

Examples of suitable mammalian expression vectors (expression vectorssuitable for use in mammalian host cells) include, but are not limitedto: recombinant viruses, nucleic acid vectors, such as plasmids,bacterial artificial chromosomes, yeast artificial chromosomes, humanartificial chromosomes, cDNA, cRNA, and polymerase chain reaction (PCR)product expression cassettes. Examples of suitable promoters for drivingexpression of a Gata4-, Mef2c-, or Tbx5-encoding nucleotide sequenceinclude, but are not limited to, retroviral long terminal repeat (LTR)elements; constitutive promoters such as CMV, HSV1-TK, SV40, EF-1α,β-actin; phosphoglycerol kinase (PGK), and inducible promoters, such asthose containing Tet-operator elements. In some cases, the mammalianexpression vector(s) encodes, in addition to exogenous Gata4, Mef2c, andTbx5 polypeptides, a marker gene that facilitates identification orselection of cells that have been transfected or infected. Examples ofmarker genes include, but are not limited to, genes encoding fluorescentproteins, e.g., enhanced green fluorescent protein, Ds-Red (DsRed:Discosoma sp. red fluorescent protein (RFP); Bevis and Glick (2002) Nat.Biotechnol. 20:83), yellow fluorescent protein, and cyanofluorescentprotein; and genes encoding proteins conferring resistance to aselection agent, e.g., a neomycin resistance gene, a puromycinresistance gene, a blasticidin resistance gene, and the like.

Examples of suitable viral vectors include, but are not limited, viralvectors based on retroviruses (including lentiviruses); adenoviruses;and adeno-associated viruses. An example of a suitable retrovirus-basedvector is a vector based on murine moloney leukemia virus (MMLV);however, other recombinant retroviruses may also be used, e.g., AvianLeukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus (MLV),Mink-Cell focus-Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus, Gibbon Abe Leukemia Virus, Mason PfizerMonkey Virus, or Rous Sarcoma Virus, see, e.g., U.S. Pat. No. 6,333,195.

In other cases, the retrovirus-based vector is a lentivirus-basedvector, (e.g., Human Immunodeficiency Virus-1 (HIV-1); SimianImmunodeficiency Virus (SIV); or Feline Immunodeficiency Virus (FIV)),See, e.g., Johnston et al., (1999), Journal of Virology, 73(6):4991-5000(FIV); Negre D et al., (2002), Current Topics in Microbiology andImmunology, 261:53-74 (SIV); Naldini et al., (1996), Science,272:263-267 (HIV).

The recombinant retrovirus may comprise a viral polypeptide (e.g.,retroviral env) to aid entry into the target cell. Such viralpolypeptides are well-established in the art, see, e.g., U.S. Pat. No.5,449,614. The viral polypeptide may be an amphotropic viralpolypeptide, e.g., amphotropic env, which aids entry into cells derivedfrom multiple species, including cells outside of the original hostspecies. The viral polypeptide may be a xenotropic viral polypeptidethat aids entry into cells outside of the original host species. In someembodiments, the viral polypeptide is an ecotropic viral polypeptide,e.g., ecotropic env, which aids entry into cells of the original hostspecies.

Examples of viral polypeptides capable of aiding entry of retrovirusesinto cells include but are not limited to: MMLV amphotropic env, MMLVecotropic env, MMLV xenotropic env, vesicular stomatitis virus-g protein(VSV-g), HIV-1 env, Gibbon Ape Leukemia Virus (GALV) env, RD114, FeLV-C,FeLV-B, MLV 10A1 env gene, and variants thereof, including chimeras. Seee.g., Yee et al., (1994), Methods Cell Biol., Pt A:99-112 (VSV-G); U.S.Pat. No. 5,449,614. In some cases, the viral polypeptide is geneticallymodified to promote expression or enhanced binding to a receptor.

In general, a recombinant virus is produced by introducing a viral DNAor RNA construct into a producer cell. In some cases, the producer celldoes not express exogenous genes. In other cases, the producer cell is a“packaging cell” comprising one or more exogenous genes, e.g., genesencoding one or more gag, pol, or env polypeptides and/or one or moreretroviral gag, pol, or env polypeptides. The retroviral packaging cellmay comprise a gene encoding a viral polypeptide, e.g., VSV-g that aidsentry into target cells. In some cases, the packaging cell comprisesgenes encoding one or more lentiviral proteins, e.g., gag, pol, env,vpr, vpu, vpx, vif, tat, rev, or nef. In some cases, the packaging cellcomprises genes encoding adenovirus proteins such as E1A or E1B or otheradenoviral proteins. For example, proteins supplied by packaging cellsmay be retrovirus-derived proteins such as gag, pol, and env;lentivirus-derived proteins such as gag, pol, env, vpr, vpu, vpx, vif,tat, rev, and nef; and adenovirus-derived proteins such as E1A and E1B.In many examples, the packaging cells supply proteins derived from avirus that differs from the virus from which the viral vector derives.

Packaging cell lines include but are not limited to anyeasily-transfectable cell line. Packaging cell lines can be based on293T cells, NIH3T3, COS or HeLa cell lines. Packaging cells are oftenused to package virus vector plasmids deficient in at least one geneencoding a protein required for virus packaging. Any cells that cansupply a protein or polypeptide lacking from the proteins encoded bysuch virus vector plasmid may be used as packaging cells. Examples ofpackaging cell lines include but are not limited to: Platinum-E(Plat-E); Platinum-A (Plat-A); BOSC 23 (ATCC CRL 11554); and Bing (ATCCCRL 11270), see, e.g., Morita et al., (2000), Gene Therapy, 7:1063-1066;Onishi et al., (1996), Experimental Hematology, 24:324-329; U.S. Pat.No. 6,995,009. Commercial packaging lines are also useful, e.g.,Ampho-Pak 293 cell line, Eco-Pak 2-293 cell line, RetroPack PT67 cellline, and Retro-X Universal Packaging System (all available fromClontech).

The retroviral construct may be derived from a range of retroviruses,e.g., MMLV, HIV-1, SIV, FIV, or other retrovirus described herein. Theretroviral construct may encode all viral polypeptides necessary formore than one cycle of replication of a specific virus. In some cases,the efficiency of viral entry is improved by the addition of otherfactors or other viral polypeptides. In other cases, the viralpolypeptides encoded by the retroviral construct do not support morethan one cycle of replication, e.g., U.S. Pat. No. 6,872,528. In suchcircumstances, the addition of other factors or other viral polypeptidescan help facilitate viral entry. In an exemplary embodiment, therecombinant retrovirus is HIV-1 virus comprising a VSV-g polypeptide butnot comprising a HIV-1 env polypeptide.

The retroviral construct may comprise: a promoter, a multi-cloning site,and/or a resistance gene. Examples of promoters include but are notlimited to CMV, SV40, EF1α, β-actin; retroviral LTR promoters, andinducible promoters. The retroviral construct may also comprise apackaging signal (e.g., a packaging signal derived from the MFG vector;a psi packaging signal). Examples of some retroviral constructs known inthe art include but are not limited to: pMX, pBabeX or derivativesthereof. See e.g., Onishi et al., (1996), Experimental Hematology,24:324-329. In some cases, the retroviral construct is aself-inactivating lentiviral vector (SIN) vector, see, e.g., Miyoshi etal., (1998), J. Virol., 72(10):8150-8157. In some cases, the retroviralconstruct is LL-CG, LS-CG, CL-CG, CS-CG, CLG or MFG. Miyoshi et al.,(1998), J. Virol., 72(10):8150-8157; Onishi et al., (1996), ExperimentalHematology, 24:324-329; Riviere et al., (1995), PNAS, 92:6733-6737.Virus vector plasmids (or constructs), include: pMXs, pMxs-IB,pMXs-puro, pMXs-neo (pMXs-IB is a vector carrying theblasticidin-resistant gene in stead of the puromycin-resistant gene ofpMXs-puro) Kimatura et al., (2003), Experimental Hematology, 31:1007-1014; MFG Riviere et al., (1995), Proc. Natl. Acad. Sci. U.S.A.,92:6733-6737; pBabePuro; Morgenstern et al., (1990), Nucleic AcidsResearch, 18:3587-3596; LL-CG, CL-CG, CS-CG, CLG Miyoshi et al., (1998),Journal of Virology, 72:8150-8157 and the like as the retrovirus system,and pAdexl Kanegae et al., (1995), Nucleic Acids Research, 23:3816-3821and the like as the adenovirus system. In exemplary embodiments, theretroviral construct comprises blasticidin (e.g., pMXs-IB), puromycin(e.g., pMXs-puro, pBabePuro); or neomycin (e.g., pMXs-neo). See, e.g.,Morgenstern et al., (1990), Nucleic Acids Research, 18:3587-3596.

Methods of producing recombinant viruses from packaging cells and theiruses are well established; see, e.g., U.S. Pat. Nos. 5,834,256;6,910,434; 5,591,624; 5,817,491; 7,070,994; and 6,995,009. Many methodsbegin with the introduction of a viral construct into a packaging cellline. The viral construct may be introduced into a host fibroblast byany method known in the art, including but not limited to: a calciumphosphate method, a lipofection method (Felgner et al. (1987) Proc.Natl. Acad. Sci. U.S.A. 84:7413-7417), an electroporation method,microinjection, Fugene transfection, and the like, and any methoddescribed herein.

A nucleic acid construct can be introduced into a host cell using avariety of well known techniques, such as non-viral based transfectionof the cell. In an exemplary aspect the construct is incorporated into avector and introduced into a host cell. Introduction into the cell maybe performed by any non-viral based transfection known in the art, suchas, but not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion. Other methods of transfection include transfectionreagents such as Lipofectamine™, Dojindo Hilymax™, Fugene™, jetPEI™,Effectene™, and DreamFect™

Additional Polypeptides

In some embodiments, a subject method does not require and does notinvolve introducing into a post-natal fibroblast any of an exogenousHopx polypeptide, an exogenous Nkx2-5 polypeptide, an exogenous Hrt2polypeptide, an exogenous Pitx2 polypeptide, an exogenous Smyd1polypeptide, an exogenous Myocd polypeptide, an exogenous Baf60cpolypeptide, an exogenous Srf polypeptide, an exogenous Isl1polypeptide, an exogenous Hand2 polypeptide, or an exogenous Mesp1polypeptide. In some embodiments, a subject method does not require anddoes not involve introducing into a post-natal fibroblast a nucleicacid(s) comprising nucleotide sequences encoding any of Hopx, Nkx2-5,Hrt2, Pitx2, Smyd1, Myocd, Baf60c, Srf, Isl1, Hand2, or Mesp1. However,in other embodiments, a subject method can involve use of Gata4, Mef2c,and Tbx5; and one or more additional polypeptides that can contribute tothe reprogramming of a post-natal fibroblast directly into acardiomyocyte.

A post-natal fibroblast can be modified (by introduction into thepost-natal fibroblast of a polypeptide), or genetically modified asdescribed above, where the post-natal fibroblast is modified with aGata4 polypeptide, an Mef2c polypeptide, and a Tbx5 polypeptide, orwhere the post-natal fibroblast is genetically modified with one or morenucleic acids comprising nucleotide sequences encoding a Gata4polypeptide, an Mef2c polypeptide, and a Tbx5 polypeptide. In someembodiments, one or more additional polypeptides (or nucleic acidscomprising nucleotide sequences encoding same) are introduced into apost-natal fibroblast, where the one or more additional polypeptides isselected from Mesp1, Isl1, Myocd, Smyd1, Srf, Baf60c, Hand2, Hopx, Hrt2,Pitx2c, and Nkx2-5.

Thus, in some embodiments, a post-natal fibroblast is modified byintroduction into the post-natal fibroblast of a Gata4 polypeptide, anMef2c polypeptide, and a Tbx5 polypeptide; and 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or 11, of a Mesp1 polypeptide, a Isl1 polypeptide, a Myocdpolypeptide, a Smyd1 polypeptide, a Srf polypeptide, a Baf60cpolypeptide, a Hand2 polypeptide, a Hopx polypeptide, a Hrt2polypeptide, a Pitx2c polypeptide, and an Nkx2-5 polypeptide. In someembodiments, a post-natal fibroblast is genetically modified with one ormore nucleic acids comprising nucleotide sequences encoding a Gata4polypeptide, an Mef2c polypeptide, and a Tbx5 polypeptide; and 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or 11, of a Mesp1 polypeptide, a Isl1 polypeptide,a Myocd polypeptide, a Smyd1 polypeptide, a Srf polypeptide, a Baf60cpolypeptide, a Hand2 polypeptide, a Hopx polypeptide, a Hrt2polypeptide, a Pitx2c polypeptide, and an Nkx2-5 polypeptide. Thefollowing are exemplary, non-limiting combinations.

In some embodiments, a post-natal fibroblast is modified by introductioninto the post-natal fibroblast of a Gata4 polypeptide, an Mef2cpolypeptide, a Tbx5 polypeptide, a Isl1 polypeptide, an Mesp1polypeptide, a Myocd polypeptide, an Nkx2.5 polypeptide, a Smyd1polypeptide, and a Srf polypeptide. In some embodiments, a post-natalfibroblast is genetically modified with one or more nucleic acidscomprising nucleotide sequences encoding a Gata4 polypeptide, an Mef2cpolypeptide, a Tbx5 polypeptide, a Isl1 polypeptide, an Mesp1polypeptide, a Myocd polypeptide, an Nkx2.5 polypeptide, a Smyd1polypeptide, and a Srf polypeptide.

In some embodiments, a post-natal fibroblast is modified by introductioninto the post-natal fibroblast of a Gata4 polypeptide, an Mef2cpolypeptide, a Tbx5 polypeptide, an Mesp1 polypeptide, a Myocdpolypeptide, and an Nkx2.5 polypeptide. In some embodiments, apost-natal fibroblast is genetically modified with one or more nucleicacids comprising nucleotide sequences encoding a Gata4 polypeptide, anMef2c polypeptide, a Tbx5 polypeptide, an Mesp1 polypeptide, a Myocdpolypeptide, and an Nkx2.5 polypeptide.

In some embodiments, a post-natal fibroblast is modified by introductioninto the post-natal fibroblast of a Gata4 polypeptide, an Mef2cpolypeptide, a Tbx5 polypeptide, an Mesp1 polypeptide, and a Myocdpolypeptide. In some embodiments, a post-natal fibroblast is geneticallymodified with one or more nucleic acids comprising nucleotide sequencesencoding a Gata4 polypeptide, an Mef2c polypeptide, a Tbx5 polypeptide,an Mesp1 polypeptide, and a Myocd polypeptide.

In some embodiments, a post-natal fibroblast is modified by introductioninto the post-natal fibroblast of a Gata4 polypeptide, an Mef2cpolypeptide, a Tbx5 polypeptide, and an Mesp1 polypeptide. In someembodiments, a post-natal fibroblast is genetically modified with one ormore nucleic acids comprising nucleotide sequences encoding a Gata4polypeptide, an Mef2c polypeptide, a Tbx5 polypeptide, and an Mesp1polypeptide.

Amino acid sequences of Mesp1, Isl1, Myocd, Smyd1, Srf, Baf60c, Hand2,Hopx, Hrt2, Pitx2c, and Nkx2-5 polypeptides are known in the art, as arenucleotide sequences encoding the polypeptides. See, e.g., GenBankAccession Nos: 1) AAR88511.1 (Homo sapiens BAF60c; amino acid sequence),and AY450431 (nucleotide sequence encoding the AAR88511.1 amino acidsequence); 2) AAR88510.1 (Homo sapiens BAF60c; amino acid sequence), andAY450430 (nucleotide sequence encoding the AAR88510.1 amino acidsequence); 3) NP_068808 (Homo sapiens Hand2 amino acid sequence), andNM_021973 (nucleotide sequence encoding the NP_068808 amino acidsequence); 4) NP_115884.4 (Homo sapiens Hopx amino acid sequence), andNM_032495 (nucleotide sequence encoding the NP_115884.4 amino acidsequence); 5) NP_631958.1 (Homo sapiens Hopx amino acid sequence), andNM_139212 (nucleotide sequence encoding the NP_631958.1 amino acidsequence); 6) AAG31157 (Homo sapiens Hrt2 amino acid sequence), andAF311884 (nucleotide sequence encoding the AAG31157 amino acid sequence;7) NP_000316 (Homo sapiens Pitx2c amino acid sequence), and NM_000325(nucleotide sequence encoding the NP_000316 amino acid sequence); 8)NP_061140.1 (Homo sapiens Mesp1 amino acid sequence), and NM_018670.3(nucleotide sequence encoding the NP_061140.1 amino acid sequence); 9)XP_523151.2 (Pan troglodytes Mesp1 amino acid sequence), and XM_523151(nucleotide sequence encoding the XP_523151.2 amino acid sequence; 10)BAA12041.1 (Mus musculus Mesp1 amino acid sequence), and BAA12041(nucleotide sequence encoding the BAA12041.1 amino acid sequence); 11)NP_001101001.1 (Rattus norvegicus Mesp1 amino acid sequence), andNM_001107531 (nucleotide sequence encoding the NP_001101001.1 amino acidsequence; 12) NP_004378.1 (Homo sapiens NKX2-5 amino acid sequence), andNM_004387 (nucleotide sequence encoding the NP_004378.1 amino acidsequence; 13) NP_001159648.1 (Homo sapiens NKX2-5 amino acid sequence),and NM_001166176 (nucleotide sequence encoding the NP_001159648.1 aminoacid sequence; 14) NP_001159647.1 (Homo sapiens Nkx2-5 amino acidsequence), and NM_001166175 (nucleotide sequence encoding theNP_001159647.1 amino acid sequence; 15) NP_002193.2 (Homo sapiens Isl1amino acid sequence), and NM_002202 (nucleotide sequence encoding theNP_002193.2 amino acid sequence); 16) NP_059035 (Rattus norvegicus Isl1amino acid), and NM_017339 (nucleotide sequence encoding the NP_059035amino acid sequence); 17) BAC41153 (Mus musculus Isl1 amino acidsequence), and AK090263 (nucleotide sequence encoding the BAC41153 aminoacid sequence); 18) NP_001139785.1 (Homo sapiens Myocd amino acidsequence), and NM_001146313 (nucleotide sequence encoding theNP_001139785.1 amino acid sequence); 19) NP_001139784.1 (Homo sapiensMyocd amino acid sequence), and NM_001146312 (nucleotide sequenceencoding the NP_001139784.1 amino acid sequence); 20) NP_705832.1 (Homosapiens Myocd amino acid sequence), and NM_153604 (nucleotide sequenceencoding the NP_705832.1 amino acid sequence); 21) NP_938015.1 (Homosapiens Smyd1 amino acid sequence), and NM_198274 (nucleotide sequenceencoding the NP_938015.1 amino acid sequence); 22) NP_001100065.1(Rattus norvegicus Smyd1 amino acid sequence), and NM_001106595(nucleotide sequence encoding the NP_001100065.1 amino acid sequence);23) NP_001153599.1 (Mus musculus Smyd1 amino acid sequence), andNM_001160127 (nucleotide sequence encoding the NP_001153599.1 amino acidsequence); 24) NP_003122.1 (Homo sapiens Srf amino acid sequence), andNM_003131 (nucleotide sequence encoding the NP_003122.1 amino acidsequence; 25) XP_518487.2 (Pan troglodytes Srf amino acid sequence), andXM_518487 (nucleotide sequence encoding the XP_518487.2 amino acidsequence; 26) NP_001102772.1 (Rattus norvegicus Srf amino acidsequence), and NM_001109302 (nucleotide sequence encoding theNP_001102772.1 amino acid sequence).

Suitable amino acid sequences of Mesp1, Isl1, Myocd, Smyd1, Srf, Baf60c,Hand2, Hopx, Hrt2, Pitx2c, and Nkx2-5 polypeptides include amino acidsequences having at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to an amino acid sequence set forth in one of theaforementioned GenBank entries.

Suitable nucleotide sequences encoding Mesp1, Isl1, Myocd, Smyd1, Srf,Baf60c, Hand2, Hopx, Hrt2, Pitx2c, and Nkx2-5 polypeptides includenucleotide sequences having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%,nucleotide sequence identity to a nucleotide sequence set forth in oneof the aforementioned GenBank entries.

Additional Factors

A post-natal fibroblast can be modified or genetically modified asdescribed above; and can also be contacted with one or more additionalfactors which can be added to the culture system, e.g., the one or moreadditional factors can be included as additives in the culture medium.Examples of such additional factors include, but are not limited to:histone deacetylase (HDAC) inhibitors, see, e.g. Huangfu et al. (2008)Nature Biotechnol. 26:795-797; Huangfu et al. (2008) Nature Biotechnol.26: 1269-1275; DNA demethylating agents, see, e.g., Mikkelson et al(2008) Nature 454, 49-55; histone methyltransferase inhibitors, see,e.g., Shi et al. (2008) Cell Stem Cell 2:525-528; L-type calcium channelagonists, see, e.g., Shi et al. (2008) 3:568-574; Wnt3a, see, e.g.,Marson et al. (2008) Cell β4:521-533; siRNA, see, e.g., Zhao et al.(2008) Cell Stem Cell 3: 475-479.

Histone deacetylases (HDAC) are a class of enzymes that remove acetylgroups from an [epsilon]-N-acetyl lysine amino acid on a histone.Exemplary HDACs include those Class I HDAC: HDAC1, HDAC2, HDAC3, HDAC8;and Class II HDACs: HDAC4, HDAC5, HDAC6, HDAC7A, HDAC9, HDAC10. Type Imammalian HDACs include: HDAC1, HDAC2, HDAC3, HDAC8, and HDACI1. Type IImammalian HDACs include: HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC1.In some embodiments, an HDAC inhibitor selectively inhibits a Class I ora Class II HDAC.

Suitable concentrations of an HDAC inhibitor range from about 0.001 nMto about 10 mM, depending on the particular HDAC inhibitor to be used.The HDAC concentration can range from 0.01 nM to 1000 nM.

Suitable HDAC inhibitors include any agent that inhibits HDAC enzymaticactivity in deacetylation of histone. Suitable HDAC inhibitors include,but are not limited to, carboxylate HDAC inhibitors; hydroxamic acidHDAC inhibitors; peptide (e.g., cyclic tetrapeptide) HDAC inhibitors;benzamide HDAC inhibitors; electrophilic ketone HDAC inhibitors; hybridpolar HDAC inhibitors; and short chain fatty acid HDAC inhibitors.

Suitable HDAC inhibitors include trichostatin A and its analogs, forexample: trichostatin A (TSA); and trichostatin C (Koghe et al., (1998),Biochem. Pharmacol, 56:1359-1364).

Suitable peptide HDAC inhibitors include, for example: oxamflatin[(2E)-5-[3-[(phenylsulfonyl)aminophenyl]-pent-2-ene-4-inohydroxamic acid(Kim et al., (1999), Oncogene, 18:2461-2470); Trapoxin A(cyclo-(L-phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10-epoxy-decanoyl)(Kijima et al., (1993), J. Biol. Chem. 268:22429-22435); FR901228,depsipeptide((1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetrazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone)(Nakajima et al., (1998). Ex. Cell Res., 241:126-133); apicidin, cyclictetrapeptide[cyclo-(N—O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)](Darkin-Rattray et al., (1996), Proc. Natl. Acad. Sci. U.S.A.,93:13143-13147; apicidin Ia, apicidin Ib, apicidin Ic, apicidin IIa, andapicidin IIb (WO 97/11366); HC-toxin, cyclic tetrapeptide (Bosch et al.(1995), Plant Cell, 7:1941-1950); and chlamydocin (Bosch et al., supra).

Suitable HDAC inhibitors include hybrid polar compounds (HPC) based onhydroxamic acid, for example: salicyl hydroxamic acid (SBHA) (Andrews etal., (2000), International J. Parasitology, 30:761-8); suberoylanilidehydroxamic acid (SAHA) (Richon et al., (1998), Proc. Natl. Acad. Sci.U.S.A., 95: 3003-7); azelaic bishydroxamic acid (ABHA) (Andrews et al.,supra); azelaic-1-hydroxamate-9-anilide (AAHA) (Qiu et al., (2000), Mol.Biol. Cell, 11:2069-83); M-carboxy cinnamic acid bishydroxamide (CBHA)(Richon et al., supra); 6-(3-chlorophenylureido) carpoic hydroxamicacid, 3-C1-UCHA) (Richon et al., supra); MW2796 (Andrews et al., supra);MW2996 (Andrews et al., supra); the hydroxamic acid derivativeNVP-LAQ-824 (Catley et al. (2003) Blood 102:2615; and Atadja et al.(2004) Cancer Res. 64:689); and CBHA (m-carboxycinnaminic acidbishydroxamic acid).

Suitable HDAC inhibitors include short chain fatty acid (SCFA)compounds, for example: sodium butyrate (Cousens et al., (1979), J.Biol. Chem., 254:1716-23); isovalerate (McBain et al., (1997), Biochem.Pharm., 53:1357-68); valproic acid; valerate (McBain et al., supra);4-phenyl butyric acid (4-PBA) (Lea and Tulsyan, (1995), AnticancerResearch, 15:879-3); phenyl butyric acid (PB) (Wang et al., (1999),Cancer Research 59: 2766-99); propinate (McBain et al., supra);butylamide (Lea and Tulsyan, supra); isobutylamide (Lea and Tulsyan,supra); phenyl acetate (Lea and Tulsyan, supra); 3-bromopropionate (Leaand Tulsyan, supra); tributyrin (Guan et al., (2000), Cancer Research,60:749-55); arginine butyrate; isobutyl amide; and valproate.

Suitable HDAC inhibitors include benzamide derivatives, for example:MS-275[N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzam-ide](Saito et al., (1999), Proc. Natl. Acad. Sci. U.S.A., 96:4592-7); and a3′-amino derivative of MS-275 (Saito et al., supra); and CI-994.

Additional suitable HDAC inhibitors include: BML-210(N-(2-aminophenyl)-N′-phenyl-octanediamide); Depudecin (e.g.,(−)-Depudecin); Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide);Scriptaid; Suramin Sodium; pivaloyloxymethyl butyrate (Pivanex, AN-9),Trapoxin B; CI-994 (i.e., N-acetyl dinaline); MGCD0103(N-(2-Aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide);JNJ16241199 (R-306465; see, e.g., Arts et al. (2007) Br. J. Cancer97:1344); Tubacin; A-161906; proxamide; oxamflatin; 3-C1-UCHA(6-(3-chlorophenylureido)caproic hydroxamic acid); and AOE(2-amino-8-oxo-9,10-epoxydecanoic acid).

Suitable DNA methylation inhibitors are inhibitors of DNAmethyltransferase, and include, but are not limited to,5-deoxy-azacytidine (DAC); 5-azacytidine (5-aza-CR) (Vidaza);5-aza-2′-deoxycytidine (5-aza-CdR; decitabine);1-[beta]-D-arabinofuranosyl-5-azacytosine; dihydro-5-azacytidine;zebularine ((1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one);Sinefungin (e.g., InSolution™ Sinefungin), and 5-fluoro-2′-deoxycyticine(FdCyd). Examples of suitable non-nucleoside DNA methyltransferseinhibitors (e.g., other than procaine) include:(−)-epigallocatechin-3-gallate (EGCG); hydralazine; procainamide;psammaplin A(N,N″-(dithiodi-2,1-ethanediyl)bis[3-bromo-4-hydroxy-a-(hydroxyimino)-benzenepropanamide);and RG 108(2-(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl)propionicacid).

Suitable histone methyltransferase (HMT) inhibitors include, but are notlimited to, SC-202651(2-(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-(1-(phenylmethyl)-4-piperidinyl)-4-quinazolinamine);chaetocin (Grainer et al. (2005) Nature Chem. Biol. 1:143); BIX-01294(2-(Hexahydro-4-methyl-1H-1,4-diazepin-1-yl)-6,7-dimethoxy-N-[1-(phenylmethyl)-4-piperidinyl]-4-quinazolinaminetrihydrochloride); 3-deazaneplanocin (Glazer et al. (1986) BBRC135:688); and the like.

Genetically Modified Host Cells

The present disclosure provides genetically modified host cells,including isolated genetically modified host cells, where a subjectgenetically modified host cell comprises (has been genetically modifiedwith) one or more exogenous nucleic acids comprising nucleotidesequences encoding one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf polypeptides. In some embodiments, asubject genetically modified host cell is in vitro. In some embodiments,a subject genetically modified host cell is a human cell or is derivedfrom a human cell. In some embodiments, a subject genetically modifiedhost cell is a rodent cell or is derived from a rodent cell. The presentdisclosure further provides progeny of a subject genetically modifiedhost cell, where the progeny can comprise the same exogenous nucleicacid as the subject genetically modified host cell from which it wasderived. The present disclosure further provides a compositioncomprising a subject genetically modified host cell.

The present disclosure provides genetically modified host cells,including isolated genetically modified host cells, where a subjectgenetically modified host cell comprises (has been genetically modifiedwith) one or more exogenous nucleic acids comprising nucleotidesequences encoding Gata4, Mef2c, and Tbx5 polypeptides. In someembodiments, a subject genetically modified host cell is in vitro. Insome embodiments, a subject genetically modified host cell is a humancell or is derived from a human cell. In some embodiments, a subjectgenetically modified host cell is a rodent cell or is derived from arodent cell. The present disclosure further provides progeny of asubject genetically modified host cell, where the progeny can comprisethe same exogenous nucleic acid as the subject genetically modified hostcell from which it was derived. The present disclosure further providesa composition comprising a subject genetically modified host cell.

Genetically Modified Post-Natal Fibroblasts

In some embodiments, a subject genetically modified host cell is agenetically modified post-natal fibroblast. Thus, the present disclosureprovides a genetically modified post-natal fibroblast that comprises(has been genetically modified with) one or more exogenous nucleic acidscomprising nucleotide sequences encoding one or more of Gata4, Mef2c,Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf polypeptides, or asubset (e.g., Gata4, Mef2c, and Tbx5 polypeptides). In some embodiments,a subject genetically modified post-natal fibroblast is in vitro. Insome embodiments, a subject genetically modified post-natal fibroblastis a human cell or is derived from a human cell. In some embodiments, asubject genetically modified post-natal fibroblast is a rodent cell oris derived from a rodent cell. The present disclosure further providesprogeny of a subject genetically modified post-natal fibroblast, wherethe progeny can comprise the same exogenous nucleic acid as the subjectgenetically modified post-natal fibroblast from which it was derived.The present disclosure further provides a composition comprising asubject genetically modified post-natal fibroblast.

Genetically Modified Induced Cardiomyocytes

The present disclosure further provides cardiomyocytes (“inducedcardiomyocytes”) derived from a subject genetically modified host cell.Because a subject induced cardiomyocyte is derived from a subjectgenetically modified post-natal fibroblast, a subject inducedcardiomyocyte is also genetically modified. Thus, the present disclosureprovides a genetically modified cardiomyocyte that comprises one or moreexogenous nucleic acids comprising nucleotide sequences encoding one ormore of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides, or a subset (e.g., Gata4, Mef2c, and Tbx5 polypeptides).In some embodiments, a subject genetically modified cardiomyocyte is invitro. In some embodiments, a subject genetically modified cardiomyocyteis a human cell or is derived from a human cell. In some embodiments, asubject genetically modified cardiomyocyte is a rodent cell or isderived from a rodent cell. The present disclosure further providesprogeny of a subject genetically modified cardiomyocyte, where theprogeny can comprise the same exogenous nucleic acid as the subjectgenetically modified cardiomyocyte from which it was derived. Thepresent disclosure further provides a composition comprising a subjectgenetically modified cardiomyocyte.

Compositions

The present disclosure provides a composition comprising a subjectgenetically modified host cell (e.g., a subject genetically modifiedpost-natal fibroblast; progeny of a subject genetically modifiedpost-natal fibroblast; a subject induced cardiomyocyte; progeny of asubject induced cardiomyocyte). A subject composition comprises asubject genetically modified host cell; and will in some embodimentscomprise one or more further components, which components are selectedbased in part on the intended use of the genetically modified host cell.Suitable components include, but are not limited to, salts; buffers;stabilizers; protease-inhibiting agents; cell membrane- and/or cellwall-preserving compounds, e.g., glycerol, dimethylsulfoxide, etc.;nutritional media appropriate to the cell; and the like.

In some embodiments, a subject composition comprises a subjectgenetically modified host cell and a matrix (a “subject geneticallymodified cell/matrix composition”), where a subject genetically modifiedhost cell is associated with the matrix. The term “matrix” refers to anysuitable carrier material to which the genetically modified cells areable to attach themselves or adhere in order to form a cell composite.In some embodiments, the matrix or carrier material is present alreadyin a three-dimensional form desired for later application. For example,bovine pericardial tissue is used as matrix which is crosslinked withcollagen, decellularized and photofixed.

For example, a matrix (also referred to as a “biocompatible substrate”)is a material that is suitable for implantation into a subject. Abiocompatible substrate does not cause toxic or injurious effects onceimplanted in the subject. In one embodiment, the biocompatible substrateis a polymer with a surface that can be shaped into the desiredstructure that requires repairing or replacing. The polymer can also beshaped into a part of a structure that requires repairing or replacing.The biocompatible substrate can provide the supportive framework thatallows cells to attach to it and grow on it.

Suitable matrix components include, e.g., collagen; gelatin; fibrin;fibrinogen; laminin; a glycosaminoglycan; elastin; hyaluronic acid; aproteoglycan; a glycan; poly(lactic acid); poly(vinyl alcohol);poly(vinyl pyrrolidone); poly(ethylene oxide); cellulose; a cellulosederivative; starch; a starch derivative; poly(caprolactone);poly(hydroxy butyric acid); mucin; and the like. In some embodiments,the matrix comprises one or more of collagen, gelatin, fibrin,fibrinogen, laminin, and elastin; and can further comprise anon-proteinaceous polymer, e.g., can further comprise one or more ofpoly(lactic acid), poly(vinyl alcohol), poly(vinyl pyrrolidone),poly(ethylene oxide), poly(caprolactone), poly(hydroxy butyric acid),cellulose, a cellulose derivative, starch, and a starch derivative. Insome embodiments, the matrix comprises one or more of collagen, gelatin,fibrin, fibrinogen, laminin, and elastin; and can further comprisehyaluronic acid, a proteoglycan, a glycosaminoglycan, or a glycan. Wherethe matrix comprises collagen, the collagen can comprise type Icollagen, type II collagen, type III collagen, type V collagen, type XIcollagen, and combinations thereof.

The matrix can be a hydrogel. A suitable hydrogel is a polymer of two ormore monomers, e.g., a homopolymer or a heteropolymer comprisingmultiple monomers. Suitable hydrogel monomers include the following:lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate(HEMA), ethyl methacrylate (EMA), propylene glycol methacrylate (PEMA),acrylamide (AAM), N-vinylpyrrolidone, methyl methacrylate (MMA),glycidyl methacrylate (GDMA), glycol methacrylate (GMA), ethyleneglycol, fumaric acid, and the like. Common cross linking agents includetetraethylene glycol dimethacrylate (TEGDMA) andN,N′-methylenebisacrylamide. The hydrogel can be homopolymeric, or cancomprise co-polymers of two or more of the aforementioned polymers.Exemplary hydrogels include, but are not limited to, a copolymer ofpoly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO); Pluronic™F-127 (a difunctional block copolymer of PEO and PPO of the nominalformula EO₁₀₀-PO₆₅-EO₁₀₀, where EO is ethylene oxide and PO is propyleneoxide); poloxamer 407 (a tri-block copolymer consisting of a centralblock of poly(propylene glycol) flanked by two hydrophilic blocks ofpoly(ethylene glycol)); a poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide) co-polymer with a nominal molecular weightof 12,500 Daltons and a PEO:PPO ratio of 2:1); apoly(N-isopropylacrylamide)-base hydrogel (a PNIPAAm-based hydrogel); aPNIPAAm-acrylic acid co-polymer (PNIPAAm-co-AAc); poly(2-hydroxyethylmethacrylate); poly(vinyl pyrrolidone); and the like.

A subject genetically modified cell/matrix composition can furthercomprise one or more additional components, where suitable additionalcomponents include, e.g., a growth factor; an antioxidant; a nutritionaltransporter (e.g., transferrin); a polyamine (e.g., glutathione,spermidine, etc.); and the like.

The cell density in a subject genetically modified cell/matrixcomposition can range from about 10² cells/mm³ to about 10⁹ cells/mm³,e.g., from about 10² cells/mm³ to about 10⁴ cells/mm³, from about 10⁴cells/mm³ to about 10⁶ cells/mm³, from about 10⁶ cells/mm³ to about 10⁷cells/mm³, from about 10⁷ cells/mm³ to about 10⁸ cells/mm³, or fromabout 10⁸ cells/mm³ to about 10⁹ cells/mm³.

The matrix can take any of a variety of forms, or can be relativelyamorphous. For example, the matrix can be in the form of a sheet, acylinder, a sphere, etc.

Implantable Devices

The present disclosure provides an implantable device (such as anintravascular stent, a scaffold, a graft (e.g., an aortic graft), anartificial heart valve, a coronary shunt, a pacemaker electrode, anendocardial lead, etc.) that comprises a reprogramming compositioncomprising one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1,Myocd, Smyd1, and Srf polypeptides. The present disclosure furtherprovides an implantable device that comprises a reprogrammingcomposition comprising one or more nucleic acids comprising nucleotidesequences encoding one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5,Isl-1, Myocd, Smyd1, and Srf polypeptides. The reprogramming composition(comprising one or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1,Myocd, Smyd1, and Srf polypeptides, or comprising one or more nucleicacids comprising nucleotide sequences encoding one or more of Gata4,Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf polypeptides)can be coated onto a surface of the implantable device, or can becontained within a reservoir in the implantable device. Where thereprogramming composition is contained within a reservoir in theimplantable device, the reservoir is structured so as to allow thereprogramming composition to elute from the device.

The present disclosure provides an implantable device (such as anintravascular stent, a scaffold, a graft (e.g., an aortic graft), anartificial heart valve, a coronary shunt, a pacemaker electrode, anendocardial lead, etc.) that comprises a reprogramming compositioncomprising a Gata4 polypeptide, a Mef2c polypeptide, and a Tbx5polypeptide. The present disclosure further provides an implantabledevice that comprises a reprogramming composition comprising one or morenucleic acids comprising nucleotide sequences encoding Gata4, Mef2c, andTbx5 polypeptides. The reprogramming composition (comprising Gata4,Mef2c, and Tbx5 polypeptides, or comprising one or more nucleic acidscomprising nucleotide sequences encoding Gata4, Mef2c, and Tbx5polypeptides) can be coated onto a surface of the implantable device, orcan be contained within a reservoir in the implantable device. Where thereprogramming composition is contained within a reservoir in theimplantable device, the reservoir is structured so as to allow thereprogramming composition to elute from the device.

When the implantable device is at a site in an individual, the nucleicacids or the polypeptides in the reprogramming composition leave theimplantable device, and the polypeptides or the nucleic acids enter intoa fibroblast at or near the site of the implantable device. Thus, asubject implantable device, when implanted in an individual, can providefor introduction of reprogramming factors, or nucleic acids encodingsame, into a fibroblast at or near the site of implant, and can therebyprovide for reprogramming of the fibroblast into a cardiomyocyte. Forexample, where a subject implantable device is a stent, the stent can beimplanted into a coronary artery, where the reprogramming factors, ornucleic acids encoding same, elute from the stent, enter fibroblasts inthe coronary vascular bed, and reprogram the fibroblasts intocardiomyocytes.

The present disclosure provides a stent comprising a reprogrammingcomposition. Intravascular stents include, e.g., self-expandable stents,balloon-expandable stents, and stent-grafts.

In some instances, a subject implantable device comprises: areprogramming composition incorporated within a first polymeric material(a first layer) that is affixed to the surface of an implantable device(e.g., a stent); and a second polymeric material (e.g., a barrier layer)is affixed to the first polymeric material, where the second polymericmaterial controls the elution rate of the polypeptides or the nucleicacids present in the reprogramming composition. As an example, the firstpolymeric material can comprise a fluoropolymer; and the secondpolymeric material can comprise an acrylic.

In some instances, a subject implantable device comprises: areprogramming composition incorporated into a polymeric layer (a firstlayer) that is coated onto a surface of an implantable device (e.g., astent); and a barrier layer over at least a portion of the polymericlayer to reduce the rate of release of the polypeptides or nucleic acidscontained within the reprogramming composition from the implantabledevice. The polymeric layer can comprise poly(methylmethacrylate) orpoly(butylmethacrylate), and can further include poly(ethylene co-vinylacetate). The barrier can comprise a polymer or an inorganic material.

Suitable polymer materials for first layer include, but are not limitedto, polyurethanes, polyesterurethanes, silicone, fluoropolymers,ethylene vinyl acetate, polyethylene, polypropylene, polycarbonates,trimethylenecarbonate, polyphosphazene, polyhydroxybutyrate,polyhydroxyvalerate, polydioxanone, polyiminocarbonates,polyorthoesters, ethylene vinyl alcohol copolymer, L-polylactide,D,L-polylactide, polyglycolide, polycaprolactone, copolymers of lactideand glycolide, polymethylmethacrylate, poly(n-butyl)methacrylate,polyacrylates, polymethacrylates, elastomers, and mixtures thereof.

Representative elastomers include, but are not limited to, athermoplastic elastomer material, polyether-amide thermoplasticelastomer, fluoroelastomers, fluorosilicone elastomer, sytrene-butadienerubber, butadiene-styrene rubber, polyisoprene, neoprene(polychloroprene), ethylene-propylene elastomer, chloro-sulfonatedpolyethylene elastomer, butyl rubber, polysulfide elastomer,polyacrylate elastomer, nitrile, rubber, polyester, styrene, ethylene,propylene, butadiene and isoprene, polyester thermoplastic elastomer,and mixtures thereof.

The barrier layer is biocompatible (i.e., its presence does not elicitan adverse response from the body). The barrier layer can have athickness ranging from about 50 angstroms to about 20,000 angstroms. Thebarrier can comprise mostly inorganic material. However, some organiccompounds (e.g., polyacrylonitrile, polyvinylidene chloride, nylon 6-6,perfluoropolymers, polyethylene terephthalate, polyethylene2,6-napthalene dicarboxylate, and polycarbonate) may be incorporated inthe barrier. Suitable inorganic materials for use within the barrierinclude, but are not limited to, inorganic elements, such as pure metalsincluding aluminum, chromium, gold, hafnium, iridium, niobium,palladium, platinum, tantalum, titanium, tungsten, zirconium, and alloysof these metals, and inorganic compounds, such as inorganic silicides,oxides, nitrides, and carbides. Generally, the solubility of the drug inthe material of the barrier is significantly less than the solubility ofthe drug in the polymer carrier. Also, generally, the diffusivity of thedrug in the material of the barrier is significantly lower than thediffusivity of the drug in the polymer carrier.

The barrier may or may not be biodegradable (i.e., capable of beingbroken down into harmless compounds by the action of the body). Whilenon-biodegradable barrier materials may be used, some biodegradablematerials may be used as barriers. For example, calcium phosphates suchas hydroxyapatite, carbonated hydroxyapatite, tricalcium phosphate,beta-tricalcium phosphate, octacalcium phosphate, amorphous calciumphosphate, and calcium orthophosphate may be used. Certain calcium saltssuch as calcium phosphate (plaster of paris) may also be used. Thebiodegradability of the barrier may act as an additional mechanism forcontrolling drug release from the underlying first layer.

Methods of affixing the first layer onto the surface of an implantabledevice, and methods of affixing a barrier layer on the first layer, areknown in the art. See, e.g., U.S. Pat. Nos. 7,695,731 and 7,691,401.

As noted above, in some embodiments, a subject implantable devicecomprises a reservoir comprising a reprogramming composition. Forexample, in some embodiments, a subject implantable device has at leastone surface for contacting a bodily tissue, organ or fluid, where theimplantable device comprises: a substrate having a contacting surface;and a drug-eluting coating on at least a portion of the contactingsurface, where the coating is comprised of a polymer having zeolitesdispersed through the polymer, and where a porous structure of thezeolites includes reservoirs containing a release agent and areprogramming composition. The release agent prevents the therapeuticmaterial from exiting the reservoir until a triggering condition is met.A triggering condition can be contact of the release agent with a bodilyfluid; a change in pH proximate to the release agent; and the like.

Biodegradable polymers, suitable for use alone or in combination,include, but are not limited to: poly(α-hydroxy acids), such as,polycapro lactone (PCL), poly(lactide-co-glycolide) (PLGA), polylactide(PLA), and polyglycolide (PGA), and combinations and blends thereofabove at different ratios to fine-tune release rates, PLGA-PEG(polyethylene glycol), PLA-PEG, PLA-PEG-PLA, polyanhydrides,trimethylene carbonates, polyorthoesters, polyaspirins,polyphosphagenes, and tyrozine polycarbonates; natural and synthetichydrogel materials, e.g., collagen, starch, chitosans, gelatin,alginates, dextrans, vinylpyrrolidone, polyvinyl alcohol (PVA),PVA-g-PLGA, PEGT-PBT copolymer (polyactive), methacrylates,poly(N-isopropylacrylamide), PEO-PPO-PEO (pluronics), PEO-PPO-PAAcopolymers, and PLGA-PEO-PLGA. Polymer matrices according to embodimentsof the present invention may include any of the following biostablepolymers, alone or in combination: polyurethanes,polymethylmethacrylates copolymers, polyvinyl acetate (PVA), polyamides,and copolymers of polyurethane and silicone.

Reprogramming Composition

The present disclosure provides reprogramming compositions.

In some embodiments, a subject reprogramming composition compriseseither: 1) a mixture of two or more of Gata4, Mef2c, Tbx5, Mesp1,Nkx2-5, Isl-1, Myocd, Smyd1, and Srf polypeptides; or 2) one or morenucleic acids comprising nucleotide sequences encoding two or more ofGata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides. The reprogramming composition can comprise, in addition tothe polypeptides or the nucleic acids, one or more of: a salt, e.g.,NaCl, MgCl, KCl, MgSO₄, etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; glycerol; and the like.

In some embodiments, a subject reprogramming composition compriseseither: 1) a mixture of Gata4, Mef2c, and Tbx5 polypeptides; or 2) oneor more nucleic acids comprising nucleotide sequences encoding Gata4,Mef2c, and Tbx5 polypeptides. The reprogramming composition cancomprise, in addition to the polypeptides or the nucleic acids, one ormore of: a salt, e.g., NaCl, MgCl, KCl, MgSO₄, etc.; a buffering agent,e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonicacid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; glycerol; and the like.

A subject reprogramming composition can be included in a subjectimplantable device, as described above. A subject reprogrammingcomposition can be administered directly into an individual. A subjectreprogramming composition is useful for reprogramming a post-natalfibroblast into a cardiomyocyte, which reprogramming can be carried outin vitro or in vivo. Reprogramming a post-natal fibroblast into acardiomyocyte can be used to treat various cardiac disorders, asdescribed below.

A subject reprogramming composition can include a pharmaceuticallyacceptable excipient. Suitable excipient vehicles are, for example,water, saline, dextrose, glycerol, ethanol, or the like, andcombinations thereof. In addition, if desired, the vehicle may containminor amounts of auxiliary substances such as wetting or emulsifyingagents or pH buffering agents. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in the art. See,e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., 17th edition, 1985. The composition or formulation to beadministered will, in any event, contain a quantity of a subjectantibody adequate to achieve the desired state in the subject beingtreated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In some embodiments, a subject reprogramming composition is formulatedas a controlled release formulation. Sustained-release preparations maybe prepared using methods well known in the art. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody in which the matrices arein the form of shaped articles, e.g. films or microcapsules. Examples ofsustained-release matrices include polyesters, copolymers of L-glutamicacid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,hydrogels, polylactides, degradable lactic acid-glycolic acid copolymersand poly-D-(−)-3-hydroxybutyric acid. Possible loss of biologicalactivity and possible changes in activity of a polypeptide or a nucleicacid comprised in sustained-release preparations may be prevented byusing appropriate additives, by controlling moisture content and bydeveloping specific polymer matrix compositions.

A subject reprogramming composition can further comprise one or moretherapeutic agents. Therapeutic agents that can be included in a subjectreprogramming composition can include, e.g., digitalis, a statin, ananti-platelet agent, an anti-coagulant, a calcium channel blocker, anangiotensin-converting enzyme inhibitor, a vasodilator, an angiotensinII receptor blocker, a beta blocker, and the like.

Utility

A subject method of reprogramming a post-natal fibroblast is useful forgenerating a population of induced cardiomyocytes, which inducedcardiomyocytes can be used in analytical assays, for generatingartificial heart tissue, and in treatment methods.

Analytical Assays

A subject method can be used to generate cardiomyocytes for analyticalassays. Analytical assays include, e.g., introduction of thecardiomyocytes into a non-human animal model of a disease (e.g., acardiac disease) to determine efficacy of the cardiomyocytes in thetreatment of the disease; use of the cardiomyocytes in screening methodsto identify candidate agents suitable for use in treating cardiacdisorders; and the like. In some cases, a cardiomyocyte generated usinga subject method can be used to assess the toxicity of a test agent orfor drug optimization.

Animal Models

In some embodiments, a cardiomyocyte generated using a subject methodcan be introduced into a non-human animal model of a cardiac disorder,and the effect of the cardiomyocyte on ameliorating the disorder can betested in the non-human animal model (e.g., a rodent model such as a ratmodel, a guinea pig model, a mouse model, etc.; a non-human primatemodel; a lagomorph model; and the like). For example, the effect of acardiomyocyte generated using a subject method on a cardiac disorder ina non-human animal model of the disorder can be tested by introducingthe cardiomyocyte into, near, or around diseased cardiac tissue in thenon-human animal model; and the effect, if any, of the introducedcardiomyocyte on cardiac function can be assessed. Methods of assessingcardiac function are well known in the art; and any such method can beused.

Drug/Agent Screening or Identification

Cardiomyocytes generated using a subject method may be used to screenfor drugs or test agents (e.g., solvents, small molecule drugs,peptides, oligonucleotides) or environmental conditions (e.g., cultureconditions or manipulation) that affect the characteristics of suchcells and/or their various progeny. See, e.g., U.S. Pat. No. 7,425,448.Drugs or test agents may be individual small molecules of choice (e.g.,a lead compound from a previous drug screen) or in some cases, the drugsor test agents to be screened come from a combinatorial library, e.g., acollection of diverse chemical compounds generated by either chemicalsynthesis or biological synthesis by combining a number of chemical“building blocks.” For example, a linear combinatorial chemical librarysuch as a polypeptide library is formed by combining a set of aminoacids in every possible way for a given compound length (e.g., thenumber of amino acids in a polypeptide compound). Millions of testagents (e.g., chemical compounds) can be synthesized through suchcombinatorial mixing of chemical building blocks. Indeed, theoretically,the systematic, combinatorial mixing of 100 interchangeable chemicalbuilding blocks results in the synthesis of 100 million tetramericcompounds or 10 billion pentameric compounds. See, e.g., Gallop et al.(1994), J. Med. Chem. 37(9), 1233. Preparation and screening ofcombinatorial chemical libraries are well known in the art.Combinatorial chemical libraries include, but are not limited to:diversomers such as hydantoins, benzodiazepines, and dipeptides, asdescribed in, e.g., Hobbs et al. (1993), Proc. Natl. Acad. Sci. U.S.A.90, 6909; analogous organic syntheses of small compound libraries, asdescribed in Chen et al. (1994), J. Amer. Chem. Soc., 116: 2661;Oligocarbamates, as described in Cho, et al. (1993), Science 261, 1303;peptidyl phosphonates, as described in Campbell et al. (1994), J. Org.Chem., 59: 658; and small organic molecule libraries containing, e.g.,thiazolidinones and metathiazanones (U.S. Pat. No. 5,549,974),pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134), benzodiazepines(U.S. Pat. No. 5,288,514).

Numerous combinatorial libraries are commercially available from, e.g.,ComGenex (Princeton, N.J.); Asinex (Moscow, Russia); Tripos, Inc. (St.Louis, Mo.); ChemStar, Ltd. (Moscow, Russia); 3D Pharmaceuticals (Exton,Pa.); and Martek Biosciences (Columbia, Md.).

In some embodiments, a cardiomyocyte generated using a subject method iscontacted with a test agent, and the effect, if any, of the test agenton a biological activity of the cardiomyocyte is assessed, where a testagent that has an effect on a biological activity of the cardiomyocyteis a candidate agent for treating a cardiac disorder or condition. Forexample, a test agent of interest is one that increases a biologicalactivity of the cardiomyocyte by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 40%, at least about 50%, at least about 75%,at least about 2-fold, at least about 2.5-fold, at least about 5-fold,at least about 10-fold, or more than 10-fold, compared to the biologicalactivity in the absence of the test agent. A test agent of interest is acandidate agent for treating a cardiac disorder or condition. In someembodiments, the contacting is carried out in vitro. In otherembodiments, the contacting is carried out in vivo, e.g, in a non-humananimal.

A “biological activity” includes, e.g., one or more of marker expression(e.g., cardiomyocyte-specific marker expression), receptor binding, ionchannel activity, contractile activity, and electrophysiologicalactivity.

For example, in some embodiments, the effect, if any, of the test agenton expression of a cardiomyocyte marker is assessed. Cardiomyocytemarkers include, e.g., cardiac troponin I (cTnI), cardiac troponin T(cTnT), sarcomeric myosin heavy chain (MHC), GATA-4, Nkx2.5, N-cadherin,β-adrenoceptor (β1-AR), a member of the MEF-2 family of transcriptionfactors, creatine kinase MB (CK-MB), myoglobin, and atrial natriureticfactor (ANF).

As another example, the effect, if any, of the test agent onelectrophysiology of the cardiomyocyte is assessed. Electrophysiologycan be studied by patch clamp analysis for cardiomyocyte-like actionpotentials. See Igelmund et al., Pflugers Arch. 437:669, 1999; Wobus etal., Ann. N.Y. Acad. Sci. 27:752, 1995; and Doevendans et al., J. Mol.Cell. Cardiol. 32:839, 2000.

As another example, in some embodiments, the effect, if any, of the testagent on ligand-gated ion channel activity is assessed. As anotherexample, in some embodiments, the effect, if any, of the test agent onvoltage-gated ion channel activity is assessed. The effect of a testagent on ion channel activity is readily assessed using standard assays,e.g., by measuring the level of an intracellular ion (e.g., Na⁺, Ca²⁺,K⁺, etc.). A change in the intracellular concentration of an ion can bedetected using an indicator appropriate to the ion whose influx iscontrolled by the channel. For example, where the ion channel is apotassium ion channel, a potassium-detecting dye is used; where the ionchannel is a calcium ion channel, a calcium-detecting dye is used; etc.

Suitable intracellular K⁺ ion-detecting dyes include, but are notlimited to, K⁺-binding benzofuran isophthalate and the like. Suitableintracellular Ca²⁺ ion-detecting dyes are listed above.

The effect of a test agent in the assays described herein can beassessed using any standard assay to observe phenotype or activity ofcardiomyocytes generated using a subject method, such as markerexpression, receptor binding, contractile activity, orelectrophysiology—either in in vitro cell culture or in vivo. See, e.g.,U.S. Pat. No. 7,425,448. For example, pharmaceutical candidates aretested for their effect on contractile activity—such as whether theyincrease or decrease the extent or frequency of contraction, using anymethods known in the art. Where an effect is observed, the concentrationof the compound can be titrated to determine the median effective dose(ED50).

Test Agent/Drug Toxicity

A cardiomyocyte generated using a subject method can be used to assessthe toxicity of a test agent, or drug, e.g., a test agent or drugdesigned to have a pharmacological effect on cardiomyocytes, e.g., atest agent or drug designed to have effects on cells other thancardiomyocytes but potentially affecting cardiomyocytes as an unintendedconsequence. In some embodiments, the disclosure provides methods forevaluating the toxic effects of a drug, test agent, or other factor, ina human or non-human (e.g., murine; lagomorph; non-human primate)subject, comprising contacting one or more cardiomyocytes generatedusing a subject method with a dose of a drug, test agent, or otherfactor and assaying the contacted cardiomyocytes for markers of toxicityor cardiotoxicity.

Any method known in the art may be used to evaluate the toxicity oradverse effects of a test agent or drug on cardiomyocytes generatedusing a subject method. Cytotoxicity or cardiotoxicity can bedetermined, e.g., by the effect on cell viability, survival, morphology,and the expression of certain markers and receptors. For example,biochemical markers of myocardial cell necrosis (e.g., cardiac troponinT and I (cTnT, cTnI)) may be used to assess drug-induced toxicity oradverse reactions in cardiomyocytes generated using a subject method,where the presence of such markers in extracellular fluid (e.g., cellculture medium) can indicate necrosis. See, e.g., Gaze and Collinson(2005) Expert Opin Drug Metab Toxicol 1(4):715-725. In another example,lactate dehydrogenase is used to assess drug-induced toxicity or adversereactions in cardiomyocytes generated using a subject method. See, e.g.,Inoue et al. (2007) AATEX 14, Special Issue: 457-462. In anotherexample, the effects of a drug on chromosomal DNA can be determined bymeasuring DNA synthesis or repair and used to assess drug-inducedtoxicity or adverse reactions in cardiomyocytes generated using asubject method. In still another example, the rate, degree, and/ortiming of [³H]-thymidine or BrdU incorporation may be evaluated toassess drug-induced toxicity or adverse reactions in cardiomyocytesgenerated using a subject method. In yet another example, evaluating therate or nature of sister chromatid exchange, determined by metaphasespread, can be used to assess drug-induced toxicity or adverse reactionsin cardiomyocytes generated using a subject method. See, e.g., A.Vickers (pp 375-410 in In vitro Methods in Pharmaceutical Research,Academic Press, 1997). In yet another example, assays to measureelectrophysiology or activity of ion-gated channels (e.g., Calcium-gatedchannels) can be used to assess drug-induced toxicity or adversereactions in cardiomyocytes generated using a subject method. In stillanother example, contractile activity (e.g., frequency of contraction)can be used to assess drug-induced toxicity or adverse reactions incardiomyocytes generated using a subject method.

In some embodiments, the present disclosure provides methods forreducing the risk of drug toxicity in a human or murine subject,comprising contacting one or more cardiomyocytes generated using asubject method with a dose of a drug, test agent, or pharmacologicalagent, assaying the contacted one or more differentiated cells fortoxicity, and prescribing or administering the pharmacological agent tothe subject if the assay is negative for toxicity in the contactedcells. In some embodiments, the present disclosure provides methods forreducing the risk of drug toxicity in a human or murine subject,comprising contacting one or more cardiomyocytes generated using asubject method with a dose of a pharmacological agent, assaying thecontacted one or more differentiated cells for toxicity, and prescribingor administering the pharmacological agent to the subject if the assayindicates a low risk or no risk for toxicity in the contacted cells.

Treatment Methods Using Cells

A subject modified or genetically modified fibroblast can be used totreat an individual in need of such treatment. Similarly, a subjectinduced cardiomyocyte can be used to treat an individual in need of suchtreatment. A subject modified or genetically modified fibroblast, or asubject induced cardiomyocyte, can be introduced into a recipientindividual (an individual in need of treatment), where introduction intothe recipient individual of a subject modified or genetically modifiedfibroblast, or a subject induced cardiomyocyte, treats a condition ordisorder in the individual. Thus, in some embodiments, a subjecttreatment method involves administering to an individual in need thereofa population of subject modified or genetically modified fibroblasts. Insome embodiments, a subject treatment method involves administering toan individual in need thereof a population of subject inducedcardiomyocytes.

In some embodiments, the present disclosure provides a method forperforming cell transplantation in a recipient individual in needthereof, the method generally involving: (i) generating an inducedcardiomyocyte from a fibroblast obtained from a donor individual,wherein the donor individual is immunocompatible with the recipientindividual; and (ii) transplanting one or more of the inducedcardiomyocytes into the recipient individual. In some embodiments, therecipient individual and the donor individual are the same individual.In some embodiments, the recipient individual and the donor individualare not the same individuals.

In some embodiments, the present disclosure provides a method forperforming cell transplantation in a recipient individual in needthereof, the method generally involving: (i) genetically modifying ahost post-natal fibroblast with one or more nucleic acids comprisingnucleotide sequences encoding Gata4, Mef2c, and Tbx5 polypeptides, wherethe host post-natal fibroblasts are obtained from a donor individual,wherein the donor individual is immunocompatible with the recipientindividual; and (ii) transplanting one or more of the geneticallymodified post-natal fibroblasts into the recipient individual. In someembodiments, the recipient individual and the donor individual are thesame individual. In some embodiments, the recipient individual and thedonor individual are not the same individuals.

In some embodiments, the present disclosure provides a method forperforming cell transplantation in a recipient individual in needthereof, the method generally involving: (i) modifying a host post-natalfibroblast by introducing into the host post-natal fibroblast one ormore of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides, or a subset (e.g., Gata4, Mef2c, and Tbx5 polypeptides),where the host post-natal fibroblasts are obtained from a donorindividual, wherein the donor individual is immunocompatible with therecipient individual; and (ii) transplanting one or more of the modifiedpost-natal fibroblasts into the recipient individual. In someembodiments, the recipient individual and the donor individual are thesame individual. In some embodiments, the recipient individual and thedonor individual are not the same individuals.

A subject method of generating induced cardiomyocytes is useful forgenerating artificial heart tissue, e.g., for implanting into amammalian subject in need thereof. In some embodiments, a subjecttreatment method involves administering to an individual in need thereofa subject artificial heart tissue.

A subject treatment method is useful for replacing damaged heart tissue(e.g., ischemic heart tissue). Where a subject method involvesintroducing (implanting) an induced cardiomyocyte into an individual,allogeneic or autologous transplantation can be carried out.

The present disclosure provides methods of treating a cardiac disorderin an individual, the method generally involving administering to anindividual in need thereof a therapeutically effective amount of: a) apopulation of induced cardiomyocytes prepared using a subject method; b)a population of genetically modified post-natal fibroblasts preparedusing a subject method; c) a population of modified post-natalfibroblasts prepared using a subject method; or d) an artificial hearttissue prepared using a subject method.

For example, in some embodiments, a subject method comprises: i)generating an induced cardiomyocyte in vitro, as described above; andii) introducing the induced cardiomyocyte into an individual in needthereof. In other embodiments, a subject method comprises: i)genetically modifying a host post-natal fibroblast with one or morenucleic acids comprising nucleotide sequences encoding one or more ofGata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides, or a subset (e.g., Gata4, Mef2c, and Tbx5 polypeptides);and ii) introducing the genetically modified post-natal fibroblasts intoan individual in need thereof.

In other embodiments, a subject method comprises: i) modifying a hostpost-natal fibroblast by introducing into the host post-natal fibroblastone or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1,and Srf polypeptides, or a subset (e.g., Gata4, Mef2c, and Tbx5polypeptides); and ii) introducing the modified post-natal fibroblastsinto an individual in need thereof.

In other embodiments, a subject method comprises: i) generatingartificial heart tissue by: a) generating an induced cardiomyocyte, asdescribed above; and b) associating the induced cardiomyocyte with amatrix, to form artificial heart tissue; and ii) introducing theartificial heart tissue into an individual in need thereof. In otherembodiments, a subject comprises: i) generating artificial heart tissueby: a) genetically modifying a host post-natal fibroblast with one ormore nucleic acids comprising nucleotide sequences encoding one or moreof Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srfpolypeptides, or a subset (e.g., Gata4, Mef2c, and Tbx5 polypeptides);and b) associating the genetically modified post-natal fibroblasts witha matrix, to form artificial heart tissue; and ii) introducing theartificial heart tissue into an individual in need thereof. In otherembodiments, a subject comprises: i) generating artificial heart tissueby: a) modifying a host post-natal fibroblast by introducing into thehost post-natal fibroblast one or more of Gata4, Mef2c, Tbx5, Mesp1,Nkx2-5, Isl-1, Myocd, Smyd1, and Srf polypeptides, or a subset (e.g.,Gata4, Mef2c, and Tbx5 polypeptides); and b) associating the modifiedpost-natal fibroblasts with a matrix, to form artificial heart tissue;and ii) introducing the artificial heart tissue into an individual inneed thereof. The artificial heart tissue can be introduced into, on, oraround existing heart tissue in the individual.

Individuals in need of treatment using a subject method and/or donorindividuals include, but are not limited to, individuals having acongenital heart defect; individuals suffering from a degenerativemuscle disease; individuals suffering from a condition that results inischemic heart tissue, e.g., individuals with coronary artery disease;and the like. In some examples, a subject method is useful to treat adegenerative muscle disease or condition, e.g., familial cardiomyopathy,dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictivecardiomyopathy, or coronary artery disease with resultant ischemiccardiomyopathy. In some examples, a subject method is useful to treatindividuals having a cardiac or cardiovascular disease or disorder,e.g., cardiovascular disease, aneurysm, angina, arrhythmia,atherosclerosis, cerebrovascular accident (stroke), cerebrovasculardisease, congenital heart disease, congestive heart failure,myocarditis, valve disease coronary, artery disease dilated, diastolicdysfunction, endocarditis, high blood pressure (hypertension),cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,coronary artery disease with resultant ischemic cardiomyopathy, mitralvalve prolapse, myocardial infarction (heart attack), or venousthromboembolism.

Individuals who are suitable for treatment with a subject method includeindividuals (e.g., mammalian subjects, such as humans; non-humanprimates; experimental non-human mammalian subjects such as mice, rats,etc.) having a cardiac condition including but limited to a conditionthat results in ischemic heart tissue, e.g., individuals with coronaryartery disease; and the like. In some examples, an individual suitablefor treatment suffers from a cardiac or cardiovascular disease orcondition, e.g., cardiovascular disease, aneurysm, angina, arrhythmia,atherosclerosis, cerebrovascular accident (stroke), cerebrovasculardisease, congenital heart disease, congestive heart failure,myocarditis, valve disease coronary, artery disease dilated, diastolicdysfunction, endocarditis, high blood pressure (hypertension),cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,coronary artery disease with resultant ischemic cardiomyopathy, mitralvalve prolapse, myocardial infarction (heart attack), or venousthromboembolism. In some examples, individuals suitable for treatmentwith a subject method include individuals who have a degenerative muscledisease, e.g., familial cardiomyopathy, dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronaryartery disease with resultant ischemic cardiomyopathy.

For administration to a mammalian host, a population of inducedcardiomyocytes, or a population of genetically modified post-natalfibroblasts, generated using a subject method can be formulated as apharmaceutical composition. A pharmaceutical composition can be asterile aqueous or non-aqueous solution, suspension or emulsion, whichadditionally comprises a physiologically acceptable carrier (i.e., anon-toxic material that does not interfere with the activity of thecardiomyocytes). Any suitable carrier known to those of ordinary skillin the art may be employed in a subject pharmaceutical composition. Theselection of a carrier will depend, in part, on the nature of thesubstance (i.e., cells or chemical compounds) being administered.Representative carriers include physiological saline solutions, gelatin,water, alcohols, natural or synthetic oils, saccharide solutions,glycols, injectable organic esters such as ethyl oleate or a combinationof such materials. Optionally, a pharmaceutical composition mayadditionally contain preservatives and/or other additives such as, forexample, antimicrobial agents, anti-oxidants, chelating agents and/orinert gases, and/or other active ingredients.

In some embodiments, an induced cardiomyocyte population, a populationof modified post-natal fibroblasts, or a population of geneticallymodified post-natal fibroblasts, is encapsulated, according to knownencapsulation technologies, including microencapsulation (see, e.g.,U.S. Pat. Nos. 4,352,883; 4,353,888; and 5,084,350). Where thecardiomyocytes, the modified post-natal fibroblasts, or the geneticallymodified post-natal fibroblasts are encapsulated, in some embodimentsthe cardiomyocytes, the modified post-natal fibroblasts, or thegenetically modified post-natal fibroblasts are encapsulated bymacroencapsulation, as described in U.S. Pat. Nos. 5,284,761; 5,158,881;4,976,859; 4,968,733; 5,800,828 and published PCT patent application WO95/05452.

In some embodiments, an induced cardiomyocyte population, a populationof modified post-natal fibroblasts, or a population of geneticallymodified post-natal fibroblasts, is present in a matrix, as describedbelow.

A unit dosage form of an induced cardiomyocyte population, a populationof modified post-natal fibroblasts, or a population of geneticallymodified post-natal fibroblasts, can contain from about 10³ cells toabout 10⁹ cells, e.g., from about 10³ cells to about 10⁴ cells, fromabout 10⁴ cells to about 10⁵ cells, from about 10⁵ cells to about 10⁶cells, from about 10⁶ cells to about 10⁷ cells, from about 10⁷ cells toabout 10⁸ cells, or from about 10⁸ cells to about 10⁹ cells.

An induced cardiomyocyte population, a population of modified post-natalfibroblasts, or a population of genetically modified post-natalfibroblasts, can be cryopreserved according to routine procedures. Forexample, cryopreservation can be carried out on from about one to tenmillion cells in “freeze” medium which can include a suitableproliferation medium, 10% BSA and 7.5% dimethylsulfoxide. Cells arecentrifuged. Growth medium is aspirated and replaced with freeze medium.Cells are resuspended as spheres. Cells are slowly frozen, by, e.g.,placing in a container at −80° C. Cells are thawed by swirling in a 37°C. bath, resuspended in fresh proliferation medium, and grown asdescribed above.

Artificial Heart Tissue

In some embodiments, a subject method comprises: a) reprogramming apopulation of post-natal fibroblasts into cardiomyocytes in vitro, e.g.,where the post-natal fibroblasts are present in a matrix, wherein apopulation of induced cardiomyocytes is generated; and b) implanting thepopulation of induced cardiomyocytes into or on an existing heart tissuein an individual. Thus, the present disclosure provides a method forgenerating artificial heart tissue in vitro; and implanting theartificial heart tissue in vivo. In some embodiments, a subject methodcomprises: a) reprogramming a population of post-natal fibroblasts intocardiomyocytes in vitro, generating a population of inducedcardiomyocytes; b) associating the induced cardiomyocytes with a matrix,forming an artificial heart tissue; and c) implanting the artificialheart tissue into or on an existing heart tissue in an individual.

The artificial heart tissue can be used for allogeneic or autologoustransplantation into an individual in need thereof. To produceartificial heart tissue, a matrix can be provided which is brought intocontact with the post-natal fibroblasts, where the post-natalfibroblasts are reprogrammed into cardiomyocytes using a subject method,as described above. This means that this matrix is transferred into asuitable vessel and a layer of the cell-containing culture medium isplaced on top (before or during the reprogramming of the post-natalfibroblasts). The term “matrix” should be understood in this connectionto mean any suitable carrier material to which the cells are able toattach themselves or adhere in order to form the corresponding cellcomposite, i.e. the artificial tissue. In some embodiments, the matrixor carrier material, respectively, is present already in athree-dimensional form desired for later application. For example,bovine pericardial tissue is used as matrix which is crosslinked withcollagen, decellularized and photofixed.

For example, a matrix (also referred to as a “biocompatible substrate”)is a material that is suitable for implantation into a subject ontowhich a cell population can be deposited. A biocompatible substrate doesnot cause toxic or injurious effects once implanted in the subject. Inone embodiment, the biocompatible substrate is a polymer with a surfacethat can be shaped into the desired structure that requires repairing orreplacing. The polymer can also be shaped into a part of a structurethat requires repairing or replacing. The biocompatible substrateprovides the supportive framework that allows cells to attach to it, andgrow on it. Cultured populations of cells can then be grown on thebiocompatible substrate, which provides the appropriate interstitialdistances required for cell-cell interaction.

Treatment Methods Using Polypeptides or Nucleic Acids

The present disclosure provides methods of reprogramming a fibroblastinto a cardiomyocyte in vivo.

In some embodiments, the methods generally involve contacting afibroblast in vivo with a reprogramming composition. As discussed above,a reprogramming composition comprises either: 1) two or more of Gata4,Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1, and Srf polypeptides;or 2) one or more nucleic acids comprising nucleotide sequences encodingtwo or more of Gata4, Mef2c, Tbx5, Mesp1, Nkx2-5, Isl-1, Myocd, Smyd1,and Srf polypeptides. As described above, a subject reprogrammingcomposition can comprise one or more additional components. A subjectreprogramming composition can be administered to an individual at ornear a treatment site, e.g., in or around the heart.

In some embodiments, the methods generally involve contacting afibroblast in vivo with a reprogramming composition. As discussed above,a reprogramming composition comprises either: 1) a mixture of Gata4,Mef2c, and Tbx5 polypeptides; or 2) one or more nucleic acids comprisingnucleotide sequences encoding Gata4, Mef2c, and Tbx5 polypeptides. Asdescribed above, a subject reprogramming composition can comprise one ormore additional components. A subject reprogramming composition can beadministered to an individual at or near a treatment site, e.g., in oraround the heart.

In some embodiments, a reprogramming composition is introduced into anindividual in need thereof in association with an implantable device.Thus, in some embodiments, the present disclosure provides methods ofreprogramming a fibroblast into a cardiomyocyte in vivo, the methodsgenerally involving introducing a subject implantable device (comprisinga subject reprogramming composition) into an individual in need thereof,where the implantable device is introduced at or near a treatment site,e.g., in or around the heart.

Individuals in need of treatment using a subject method and/or donorindividuals include, but are not limited to, individuals having acongenital heart defect; individuals suffering from a degenerativemuscle disease; individuals suffering from a condition that results inischemic heart tissue, e.g., individuals with coronary artery disease;and the like. In some examples, a subject method is useful to treat adegenerative muscle disease or condition, e.g., familial cardiomyopathy,dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictivecardiomyopathy, or coronary artery disease with resultant ischemiccardiomyopathy. In some examples, a subject method is useful to treatindividuals having a cardiac or cardiovascular disease or disorder,e.g., cardiovascular disease, aneurysm, angina, arrhythmia,atherosclerosis, cerebrovascular accident (stroke), cerebrovasculardisease, congenital heart disease, congestive heart failure,myocarditis, valve disease coronary, artery disease dilated, diastolicdysfunction, endocarditis, high blood pressure (hypertension),cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,coronary artery disease with resultant ischemic cardiomyopathy, mitralvalve prolapse, myocardial infarction (heart attack), or venousthromboembolism.

Individuals who are suitable for treatment with a subject method includeindividuals (e.g., mammalian subjects, such as humans; non-humanprimates; experimental non-human mammalian subjects such as mice, rats,etc.) having a cardiac condition including but limited to a conditionthat results in ischemic heart tissue, e.g., individuals with coronaryartery disease; and the like. In some examples, an individual suitablefor treatment suffers from a cardiac or cardiovascular disease orcondition, e.g., cardiovascular disease, aneurysm, angina, arrhythmia,atherosclerosis, cerebrovascular accident (stroke), cerebrovasculardisease, congenital heart disease, congestive heart failure,myocarditis, valve disease coronary, artery disease dilated, diastolicdysfunction, endocarditis, high blood pressure (hypertension),cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy,coronary artery disease with resultant ischemic cardiomyopathy, mitralvalve prolapse, myocardial infarction (heart attack), or venousthromboembolism. In some examples, individuals suitable for treatmentwith a subject method include individuals who have a degenerative muscledisease, e.g., familial cardiomyopathy, dilated cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronaryartery disease with resultant ischemic cardiomyopathy.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Direct Reprogramming of Cardiac Fibroblasts into FunctionalCardiomyocytes by Defined Factors

Materials and Methods

Generation of αMHC-GFP and Isl1-YFP Mice

To generate α-myosin heavy chain-green fluorescent protein (αMHC-GFP)mice, enhanced green fluorescent protein-internal ribosome bindingsite-puromycin^(R) (EGFP-IRES-Puromycin) cDNA were subcloned into theexpression vector containing α-myosin heavy chain (α-MHC) promoter(Gulick et al. (1991) J Biol Chem 266, 9180-9185). Pronuclearmicroinjection and other procedures were performed according to thestandard protocols (Ieda et al. (2007) Nat Med 13, 604-612. Thetransgene was identified by polymerase chain reaction (PCR) analysis(the forward primer, 5′-ATGACAGACAGATCCCTCCT-3′ (SEQ ID NO:11); thereverse primer, 5′-AAGTCGTGCTGCTTCATGTG-3′ (SEQ ID NO:12)). Isl1-yellowfluorescent protein (Isl1-YFP) mice were obtained by crossing Isl1-Cremice and R26R-enhanced yellow fluorescent protein (R26R-EYFP) mice(Srinivas et al. (2001) BMC Dev Biol 1, 4).

Cell Culture

For explant culture, isolated neonatal or adult mouse hearts were mincedinto small pieces less than 1 mm³ in size. The explants were plated ongelatin-coated dishes, and cultured for 7 days in explant medium(IMDM/20% FBS) (Andersen et al. (2009) Stem Cells 27, 1571-1581).Migrated cells were harvested and filtered with 40-μm cell strainers(BD) to avoid contamination of heart tissue fragments. αMHC-GFP⁻/Thy1⁺,Isl1-YFP⁻/Thy1⁺, αMHC-GFP⁻/Thy1⁺/c-kit⁻ or αMHC-GFP⁻/Thy1⁺/c-kit⁺ livecells (as defined by the lack of propidium iodine staining) wereisolated using fluorescence-activated cell sorting (FACS) Aria 2 (BDBiosciences). For conventional isolation of neonatal cardiacfibroblasts, hearts were digested with 0.1% trypsin and plated onplastic dishes (Ieda et al., (2009) Dev Cell 16:233). Attachedfibroblasts were cultured for 7 days and sorted αMHC-GFP⁻/Thy1⁺ cells.Sorted cells were cultured in DMEM/M199 medium containing 10% fetalbovine serum (FBS) at a density of 10⁴/cm². Cells were transduced byretroviruses after 24 h.

Isolation of Cardiomyocytes

To isolate cardiomyocytes, neonatal αMHC-GFP⁺ ventricles were cut intosmall pieces and digested with collagenase type II solution (Ieda etal., (2009) supra). A single-cell suspension was obtained by gentletriturating and passing through a 40-μm cell strainer. αMHC-GFP⁺ livecells were isolated by FACS Aria 2.

Molecular Cloning and Retroviral Infection

Retroviruses were generated as described (Kitamura et al., (2003) Exp.Hematol. 31:1007; Takahashi and Yamanaka, (2006) Cell 126:663). Briefly,to construct pMXs retroviral vectors, the coding regions of candidategenes were amplified by polymerase chain reaction (PCR) and subclonedinto pMXs vector. The pMXs retroviral vectors were transfected intoPlat-E cells with Fugene 6 (Roche) to generate viruses. Pool ofvirus-containing supernatants was used for transduction. After 24 h, themedium was replaced with DMEM/M199 medium and changed every 2-3 days.The pMXs-DsRed Express retrovirus infection in cardiac fibroblastsresulted in >95% transfection efficiency (Hong et al., (2009) Nature460:1132).

Mouse nucleotide sequences of Mef2c, Tbx5, and Gata4 used in theconstructs are set forth in SEQ ID NOs:23, 25, and 27, respectively.Amino acid sequences of the encoded Mef2c, Tbx5, and Gata4 polypeptidesare set forth in SEQ ID NOs:24, 26, and 28, respectively.

FACS Analyses and Sorting

For green fluorescent protein (GFP) expression analyses, cells wereharvested from cultured dishes and analyzed on a FACS Calibur (BDBiosciences) with FlowJo software. For αMHC-GFP/cTnT expression, cellswere fixed with 4% PFA for 15 min, permealized with Saponin, and stainedwith anti-cTnT and anti-GFP antibodies, followed by secondary antibodiesconjugated with Alexa 488 and 647 (Kattman et al., (2006) Dev. Cell11:723).

For αMHC-GFP⁻/Thy1⁺ and Isl1-YFP⁻/Thy1⁺ cell sorting, cells wereincubated with PECy7-conjugated anti-Thy1 antibody (eBioscience) andsorted by FACS Aria 2 (Ieda et al., (2009) supra). ForαMHC-GFP⁻/Thy1⁺/c-kit⁻ and αMHC-GFP⁻/Thy1⁺/c-kit⁺ cell sorting,PECy7-conjugated anti-Thy1 and allophycocyanin (APC)-conjugatedanti-c-kit antibodies (BD) were used. Bone marrow cells were used as apositive control for c-kit staining. PECy7 is a conjugate ofphycoerythrin and Cy7 fluorescent dyes.

Cell Transplantation

Fibroblasts were harvested the next day after retroviral infection. Aleft thoracotomy was carried out in non-obese diabetic-severe combinedimmunodeficiency (NOD-SCID) mice, and 10⁶ cultured cells were injectedinto the left ventricle. After 1-2 weeks, the hearts were excised forimmunohistochemistry.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde for 15 min at room temperature,blocked, and incubated with primary antibodies against sarcomericα-actinin (Sigma Aldrich), vimentin (Progen), GFP (Invitrogen), Thy-1(BD Biosciences), cardiac troponin T (cTnT) (Thermo Scientific), Nppa(Chemicon), RFP (Rockland), Nkx2.5 (Santa Cruz), with secondaryantibodies conjugated with Alexa 488 or 594 (Molecular Probes), and DAPI(Invitrogen).

Histology

For immunohistochemical studies in cell-injected hearts, hearts werefixed in 0.4% paraformaldehyde overnight, embedded in OCT compound, andfrozen in liquid nitrogen (Ieda et al., (2007) supra; Ieda et al.,(2009) supra). Hearts were cut vertically in 7-μm sections to show bothventricles. Sections were stained with primary antibodies againstactinin, red fluorescent protein (RFP), green fluorescent protein (GFP),with secondary antibodies conjugated with Alexa 488 or 594, and4′,6′-diamino-2-phenylindole (DAPI). To analyze GFP expression patternin αMHC-GFP hearts, hearts were cut longitudinally and stained withactinin, GFP and vimentin.

Quantitative RT-PCR

Total RNA was isolated from cells, and quantitative reversetranscription-polymerase chain reaction (qRT-PCR) was performed on anABI 7900HT (Applied Biosystems) with TaqMan probes (Applied Biosystems):Actc1 (Mm01333821_m1), Col1a2 (Mm00483888_m1), Myh6 (Mm00440354_m1),Ryr2 (Mm00465877_m1), Gja1 (Mm00439105_m1), Tbx5 (Mm00803521_m1). ThemRNA levels were normalized by comparison to Gapdh mRNA.

Microarray Analyses

Mouse genome-wide gene expression analyses were performed usingAffymetrix Mouse Gene 1.0 ST Array. αMHC-GFP⁺ cardiomyocytes werecollected by FACS. Three-factor transduced GFP⁺ cells and GFP⁻ cellswere collected by FACS after 4 weeks of culture. Cardiac fibroblastswere also collected after 4 weeks of culture. RNA was extracted usingPicoPure RNA Isolation (Arcturus). Microarray analyses were performed intriplicate from independent biologic samples, according to the standardAffymetrix Genechip protocol. Data were analyzed using the AffymetrixPower Tool (APT, version 1.8.5). Linear models were fitted for each geneon the sample group to derive estimated group effects and theirassociated significance with the limma package (Smyth, 2004) inR/Bioconductor. Moderated t-statistics and the associated p-values werecalculated. P-values were adjusted for multiple testing by controllingfor false-discovery rate by the Benjamini-Hochberg method. Geneannotations were retrieved from Affymetrix (version Nov. 12, 2007).Differential gene expression was defined using the statistics/thresholdcombination.

Ca²⁺ Imaging

Ca²⁺ imaging was performed according to the standard protocol. Briefly,cells were labeled with Rhod-3 (Invitrogen) for 1 h at room temperature,washed, and incubated for an additional 1 h to allow de-esterificationof the dye. Rhod-3 labeled cells were analyzed by Axio Observer (Zeiss)with MiCAM02 (SciMedia).

Electrophysiology

After 4-week transduction with three factors, the electrophysiologicalactivities of induced cardiomyocytes were performed using extracellularelectrode recording with an Axopatch 700B amplifier and the pClamp9.2software (Axon Instruments). Induced cardiomyocytes were visuallyidentified by GFP expression and spontaneous contraction. Glass patchpipettes, with typical resistances of 2-4 MΩ, were directly attached onsingle GFP⁺ cells for extracellular recording in Tyrode's bath solution.

Statistical Analyses

Differences between groups were examined for statistical significanceusing Student's t-test or ANOVA. P values of <0.05 were regarded assignificant.

Results

Screening for Cardiomyocyte Inducing Factors

An assay system was developed in which the induction of maturecardiomyocytes from fibroblasts could be analyzed quantitatively byreporter-based fluorescence-activated cell sorting (FACS) (FIG. 1A). Toaccomplish this, αMHC promoter-driven EGFP-IRES-Puromycin transgenicmice (ciMHC-GFP) were generated, in which only mature cardiomyocytesexpressed the green fluorescent protein (GFP) (Gulick et al., (1991)supra). It was confirmed that only cardiomyocytes, but not other celltypes including cardiac fibroblasts, expressed GFP in the transgenicmouse hearts.

To have enough cardiac fibroblasts for FACS screening, GFP⁻ cardiacfibroblasts were obtained from neonatal αMHC-GFP hearts by explantculture. Fibroblast-like cells migrated out from the explants after 2days and were confluent after 1 week. The migrating cells did notexpress GFP, but expressed Thy1 and Vimentin, markers of cardiacfibroblasts (FIG. 1B) (Hudon-David et al., (2007) J. Mol. Cell. Cardiol.42:991; Ieda et al., (2009) supra). To avoid contamination ofcardiomyocytes, the cells were filtered by cell strainers to removeheart tissue fragments and isolated Thy1⁺/GFP⁻ cells by FACS (FIG. 1C).The purity of cardiac fibroblasts with Thy1 as a marker for FACS waspreviously shown (Ieda et al., (2009) supra). Using these procedures, nocardiomyocyte contamination was found in the fibroblast culture, andgreater than twice the number of cardiac fibroblasts could be generatedthan by conventional fibroblast isolation techniques (Ieda et al.,(2009) supra).

To select potential cardiac reprogramming factors, microarray analyseswere used to identify transcription factors and epigenetic remodelingfactors with greater expression in mouse cardiomyocytes (CM) than incardiac fibroblasts (CF) at embryonic day 12.5 (Ieda et al., (2009)supra). Among them, 13 factors were selected that exhibited severedevelopmental cardiac defects and embryonic lethality when mutated(Table).

TABLE Relative expression Relative expression Gene CM vs. CF (E12) GeneCM vs. CF (E12) Hopx 33.1 Tbx5 3.0 Nkx2-5 30.7 Srf 2.5 Hrt2 29.6 Gata42.2 Pitx2 24.1 Isl1 2.1 Smyd1 20.6 Mef2c 2.0 Myocd 7.5 Hand2 1.8 Baf60c3.9 Mesp1 ND

Table.

Transcription factors upregulated in embryonic cardiomyocytes comparedto cardiac fibroblasts by microarray are listed along with their foldenrichment (n=3). Mesp1 expression was not detected (ND) in either celltype.

The cardiovascular mesoderm-specific transcription factor Mesp1 was alsoincluded because of its cardiac transdifferentiation effect in Xenopus(David et al., (2008) Nat. Cell Biol. 10:338). Individual retroviruseswere generated, to efficiently express each gene in cardiac fibroblasts.

Thy1⁺/GFP⁻ neonatal mouse cardiac fibroblasts were transduced with amixture of retroviruses expressing all 14 factors or with DsRedretrovirus (negative control) (Hong et al., (2009) supra). No GFP⁺ cellsin cardiac fibroblasts were observed 1 week after Ds-Red retrovirusinfection or 1 week of culture without any viral infection. In contrast,transduction of all 14 factors into fibroblast cells resulted in thegeneration of a small number of GFP⁺ cells (1.7%), indicating thesuccessful activation of the cardiac-enriched αMHC gene in some cells(FIGS. 1D and E).

To determine which of the 14 factors were critical for activatingcardiac gene expression, individual factors were serially removed fromthe pool of 14. Pools lacking five factors (Baf60c, Hand2, Hopx, Hrt2,and Pitx2c) produced an increased number of GFP⁺ cells, suggesting theyare dispensable (FIGS. 1D and E). Of note, removal of Gata4 decreasedthe percentage of GFP⁺ cells to 0.5%, while removal of Pitx2c increasedit to 5%. Three further rounds of single factor withdrawal wereconducted from nine-, six-, and five-factor pools; it was found thatfour factors (Gata4, Mef2c, Mesp1, and Tbx5) were sufficient forefficient GFP⁺ cell induction from cardiac fibroblasts (FIG. 1F-H). Thecombination of these four factors dramatically increased the number offibroblasts activating the αMHC-GFP reporter to over 20% (FIG. 1I).

FIGS. 1A-I. Screening for Cardiomyocyte Inducing Factors

(A) Schematic representation of the strategy to test candidate factors.(B) Morphology and characterization of fibroblast-like cells migratingfrom αMHC-GFP heart explants. Phase contrast (left), GFP (middle), andThy-1 immunostaining (right). Insets are high-magnification views. (C)Thy-1⁺/GFP⁻ cells were FACS sorted from explant culture forreprogramming after filtration with cell strainers to remove myocytes.(D) Summary of FACS analyses for GFP⁺ cells. Effect on GFP⁺ cellinduction with 14 factors or the removal of individual factors from thepool of 14 factors (n=3). (E) FACS plots for analyses of GFP⁺ cells.GFP⁺ cells were analyzed 1 week after 14-factor transduction. The numberof GFP⁺ cells were reduced by removal of Gata4, but increased by removalof Pitx2c from 14 factors. (F-H) Effect on GFP⁺ cell induction of theremoval of individual factors from the pool of 9 (F), 6 (G), or 5 (H)factors (n=3 in each case). (I) GFP⁺ cells were induced from fibroblastsby the combination of four factors, Gata4, Mef2c, Mesp1, and Tbx5.Representative data are shown in each panel. All data are presented asmeans±SD. PI, propidium iodine. *, P<0.01; **, P<0.05 vs relativecontrol. Scale bars, 100 μm.

Gata4, Mef2c, and Tbx5 are Necessary and Sufficient for CardiomyocyteInduction

Next, expression of cardiac troponin T (cTnT), a specific sarcomericmarker of differentiated cardiomyocytes (Kattman et al., (2006) supra),was examined. It was found that 20% of GFP⁺ cells expressed cTnT 1 weekafter the four-factor transduction. Again removing individual factorsfrom the four-factor pool in transduction, it was found that Mesp1 wasdispensable for cTnT expression (FIGS. 2A and B). In contrast, cTnT⁺ orGFP⁺ cells were not observed, when either Mef2c or Tbx5 was removed.Removal of Gata4 did not significantly affect the number of GFP⁺ cells,but cTnT expression was completely abolished, suggesting Gata4 was alsorequired. The combination of two factors, Mef2c and Tbx5, induced GFPexpression but not cTnT. No other combination of two factors or singlefactor induced both GFP and cTnT expression in cardiac fibroblasts (FIG.2C). These data suggested that the combination of three factors, Gata4,Mef2c, and Tbx5, is necessary and sufficient to induce cardiac geneexpression. To confirm the screening results, cardiac fibroblasts weretransduced with three factors (Gata4, Mef2c, and Tbx5) plus Nkx2-5, acritical factor for cardiogenesis but excluded by the initial screening.Surprisingly, adding Nkx2-5 dramatically inhibited the expression of GFPand cTnT in cardiac fibroblasts, confirming the screening results (FIG.2D).

Next, immunocytochemistry was used to determine if other cardiac markerswere expressed in GFP⁺ cells. Most GFP⁺ cells induced with the threefactors expressed sarcomeric α-actinin (actinin) and had well-definedsarcomeric structures (FIGS. 2E and F). GFP⁺ cells also expressed cTnTand ANF (atrial natriuretic factor), indicating GFP⁺ cells expressedseveral cardiomyocyte-specific markers (FIG. 2F).

FIGS. 2A-F. Combination of Three Transcription Factors Induces CardiacGene Expression in Fibroblasts

(A) FACS analyses for α-MHC-GFP and cardiac Troponin T (cTnT)expression. Effects of the removal of individual factors from the poolof four factors on GFP⁺ and cTnT⁺ cell induction. Note that removal ofGata4 did not affect GFP⁺, but cTnT expression was strongly inhibited.(B) Quantitative data of GFP⁺ cells and cTnT⁺ cells in (A) (n=3). (C)Effect of the transduction of pools of three, two, and one factors onGFP⁺ and cTnT⁺ cell induction (n=3). (D) FACS plot showing that Nkx2-5inhibited reprogramming induced with three (GMT; Gata4, Mef2c and Tbx5)factors. (E) Immunofluorescent staining for GFP (green), actinin (red)and DAPI (blue). The combination of three factors, Gata4, Mef2c andTbx5, induced abundant GFP expression in cardiac fibroblasts 2 weeksafter transduction. Note that the majority of GFP⁺ cells were positivefor actinin. (F) Induced cardiomyocytes expressed several cardiacmarkers by immunocytochemistry with clear sarcomeric organization(actinin and Nppa, 2 weeks after transduction; cTnT, 4 weeks aftertransduction). Insets are high-magnification views. All data arepresented as means±SD. *, P<0.01 vs relative control. Scale bars, 100μm.

Induced Cardiomyocytes Are Directly Differentiated from CardiacFibroblasts

Next, neonatal cardiac fibroblasts were isolated by the conventionalfibroblast isolation method in which hearts were digested with trypsinand plated on plastic dishes (Ieda et al., (2009) supra). More than 80%of the cells expressed Thy1, and Thy1⁺/GFP⁻ cells were isolated by FACSto exclude cardiomyocyte contamination (FIG. 3A). Fibroblasts transducedwith Gata4/Mef2c/Tbx5, hereafter referred to as GMT, expressed GFP, cTnTand actinin after 1 week at the same level as fibroblasts isolated fromexplant cultures (FIGS. 3B and C). Similar results were obtained uponintroduction of GMT into adult cardiac fibroblasts, with full formationof sarcomeric structures (FIGS. 3D and E).

To determine if the induced cardiomyocytes were arising from asubpopulation of stem-like cells, c-kit expression (Beltrami et al.,(2003) Cell 114:763; Wu et al., (2006) Cell 127:1137) was analyzed inthe Thy1⁺/GFP⁻ cells. Most c-kit⁺ cells co-expressed Thy1, while 15% ofThy1⁺ cells expressed c-kit. GFP⁻/Thy1⁺/c-kit⁺ cells andGFP⁻/Thy1⁺/c-kit⁻ cells were isolated by FACS and transduced eachpopulation of cells with the three factors. The results showed 2-3-foldmore cardiomyocyte induction from GFP⁻/Thy1⁺/c-kit⁻ cells than fromGFP⁻/Thy1⁺/c-kit⁺ cells (FIG. 3F-H). These results suggest that most ofthe induced cardiomyocytes originated from a c-kit negative population.

Next, it was investigated whether the reprogramming of cardiacfibroblasts to differentiated cardiomyocytes was a direct event or ifthe fibroblasts first passed through a cardiac progenitor cell fatebefore further differentiation. To distinguish between these twopossibilities, Isl1-YFP mice were used, which were obtained by crossingIsl1-Cre mice and R26R-EYFP mice (Srinivas et al., (2001) supra). Isl1is an early cardiac progenitor marker that is transiently expressedbefore cardiac differentiation. If cardiomyocytes generated fromfibroblasts passed through a cardiac progenitor state, they and theirdescendents should permanently express YFP (Laugwitz et al., (2005)Nature 433:647). Isl1-YFP⁻/Thy1⁺ cells were isolated from Isl1-YFP heartexplants by FACS and transduced the cells with Gata4, Mef2c, and Tbx5.The resulting cTnT⁺ cells did not express YFP, suggesting that theinduced cardiomyocytes (iCMs) were not first reprogrammed into cardiacprogenitor cells. Moreover, these results indicated that iCMs did notoriginate from a rare population of cardiac progenitor cells, whichmight exist in neonatal hearts (FIGS. 3I and J).

FIGS. 3A-J. Induced Cardiomyocytes Originate from Differentiated CardiacFibroblasts and are Directly Reprogrammed

(A) Cardiac fibroblasts isolated by the conventional isolation method.Most cells were positive for Thy1, and Thy-1⁺/GFP⁻ cells were sorted byFACS for transduction. (B) FACS analyses for GFP and cTnT expression incardiac fibroblasts isolated in (A) one week after transduction by threefactors (GMT). (C) Immunofluorescent staining for GFP (green), actinin(red) and DAPI (blue) in the three-factor induced cardiomyocytesoriginated from (A). (D) Cardiac fibroblasts isolated from adultαMHC-GFP hearts were transduced with three factors. (E)Immunofluorescent staining for GFP, actinin and DAPI in the inducedcardiomyocytes originated from adult cardiac fibroblasts indicated in(D). (F) GFP⁻/Thy1⁺/c-kit⁺ cells and GFP⁻/Thy1⁺/c-kit⁻ cells wereisolated by FACS, and transduced with 3 factors. (G) GFP⁻/Thy1⁺/c-kit⁻cells expressed more GFP and cTnT than GFP⁻/Thy1⁺/c-kit⁺ cells bythree-factor transduction. (H) Quantitative data of GFP⁺ cells and cTnT⁺cells in (G) (n=3). (I) Isl1-YFP⁻/Thy1⁺ cells were sorted from Isl1-YFPheart explants and transduced with three factors. (J) The vast majorityof cTnT⁺ cells induced from Isl1-YFP⁻/Thy1⁺ cells were negative for YFP.All data are presented as means±SD. *, P<0.01 vs relative control. Scalebars, 100 μm.

Induced Cardiomyocytes Resemble Neonatal Cardiomyocytes in Global GeneExpression

The time course of cardiomyocyte induction was analyzed. GFP⁺ cells weredetected 3 days after induction and gradually increased in number up to20% at day 10, and were still present after 4 weeks (FIG. 4A).Importantly, the percentage of cTnT cells and the intensity of cTnTexpression in GFP⁺ cells increased significantly over time (FIGS. 4B andC). GFP⁺ cells were sorted at 7, 14, and 28 days after transduction withGMT and compared candidate gene expression with cardiac fibroblasts andneonatal cardiomyocytes. The cardiomyocyte-specific genes, Actc1(cardiac α-actin), Myh6 (α-myosin heavy chain), Ryr2 (ryanodine receptor2), and Gja1 (connexin43), were significantly upregulated in atime-dependent manner in GFP⁺ cells, but were not detected in cardiacfibroblasts by quantitative RT-PCR (qPCR). Colla2 (collagen 1a2), amarker of fibroblasts, was dramatically downregulated in GFP⁺ cells from7-day culture to the same level as in cardiomyocytes. Expression of thethree transduced genes was strongly upregulated in inducedcardiomyocytes up to 4 weeks later, suggesting they were not silenced(FIG. 4D). These data indicated that the three factors induced directconversion of cardiac fibroblasts to cardiomyocytes rapidly andefficiently, but full maturation was a slow process.

The global gene expression pattern of iCMs, neonatal cardiomyocytes, andcardiac fibroblasts was compared by mRNA microarray analyses. GFP⁺ cellsand GFP⁻ cells were sorted 28 days after GMT transduction. The inducedGFP⁺ cells were strikingly similar to neonatal cardiomyocytes, but weredistinct from GFP⁻ cells and cardiac fibroblasts in global geneexpression pattern. These results demonstrate that iCMs are highlysimilar to neonatal cardiomyocytes, indicating that the reprogrammingevent was broadly reflected in global gene expression.

FIGS. 4A-D. Gene Expression of Induced Cardiomyocytes is GloballyReprogrammed

(A) The percent of GFP⁺ cells compared to the number of plated cells(n=3). The number of GFP⁺ cells was counted by FACS sorting at each timepoint. (B) FACS analyses of cTnT expression in GFP⁺ cells. Note thatcTnT⁺ cell number and cTnT intensity were both increased over time(n=3). (C) Quantitative data of cTnT intensity in (B) (n=4). (D) Actc1,Myh6, Ryr2, Gja1, Colla2 and Tbx5 mRNA expression in cardiac fibroblasts(CF), induced cardiomyocytes (GFP⁺, 7 d, 14 d, 28 d after transduction)and neonatal cardiomyocytes (CM), determined by qPCR (n=3).

Induced Cardiomyocytes Exhibit Spontaneous Contraction

To determine if iCMs possessed the functional properties characteristicof cardiomyocytes, intracellular Ca²⁺ flux was analyzed in iCMs after 4weeks of culture. Around 30% of iCMs showed spontaneous Ca²⁺oscillations that resembled those of neonatal cardiomyocytes (FIG. 5A).Ca²⁺ oscillation frequency was variable among the cells (FIG. 5B). Inaddition, spontaneous Ca²⁺ waves were observed in iCMs, similar toneonatal cardiomyocytes (FIG. 5C).

In addition to the characteristic Ca²⁺ flux, iCMs showed spontaneouscontractile activity after 4-5 weeks in culture. Single cellextracellular recording of electrical activity in beating cells revealedtracings similar to neonatal cardiomyocytes (FIG. 5D) (Yeung et al.,2001). Thus, the reprogramming of cardiac fibroblast to iCMs wasassociated with global changes in gene expression and the functionalproperties characteristic of cardiomyocytes.

FIGS. 5A-D. Induced Cardiomyocytes Exhibit Spontaneous Ca²⁺ Flux,Electrical Activity and Beating

(A) α-MHC-GFP⁺ cells showed spontaneous Ca²⁺ oscillation. Thepseudo-color image shows Rhod-3 fluorescence intensity in cells. Smallsquares indicate the Ca²⁺ measuring areas, and the inset is ahigh-magnification view (left panel). The Rhod-3 intensity trace (rightpanel) corresponds to the left panel. The arrow indicates the time pointcorresponding to the image on the left. (B) High frequency of Ca²⁺oscillation was observed in induced cardiomyocytes. The arrow indicatesthe time point corresponding to the image on the left. (C) SpontaneousCa²⁺ waves observed in the induced cardiomyocyte. GFP and Rhod-3 at Ca²⁺max and min, are shown. The GFP⁺ cell is outlined in white dots. (D)Spontaneously contracting cells had electrical activity measured bysingle cell extracellular electrodes. Representative data are shown ineach panel (n=10).

Cardiac Fibroblasts Convert to Cardiomyocytes by Three-FactorTransduction In Vivo

Next, to investigate whether Gata4+Mef2c+Tbx5 (GMT)-transduced cardiacfibroblasts can be reprogrammed to cardiomyocytes in vivo, GFP⁻/Thy1⁺cardiac fibroblasts were harvested at day 1 after viral transduction andinjected them into non-obese diabetic-severe combined immunodeficiency(NOD-SCID) mouse hearts. GMT-infected cells did not express GFP at day 1after transduction (FIG. 4A). Cardiac fibroblasts were infected witheither the mixture of the three factors and DsRed retroviruses or DsRedretrovirus (negative control) to be readily identified by fluorescence.Cardiac fibroblasts infected with DsRed did not express actinin or GFP,confirming cardiomyocyte conversion did not happen in the negativecontrol (FIGS. 6A and B). Despite being injected into the heart only 1day after viral infection, a subset of cardiac fibroblasts transducedwith the three factors (GMT) and DsRed expressed GFP in the mouse heartwithin 2 weeks (FIG. 6B). The GFP⁺ cells expressed actinin and had clearsarcomeric structures (FIG. 6C). These results suggested that Gata4,Mef2c, and Tbx5 were sufficient to convert cardiac fibroblasts tocardiomyocytes within two weeks in vivo.

FIGS. 6A-C. Cardiac Fibroblasts Can Be Reprogrammed to Cardiomyocytes InVivo

(A) DsRed infected cardiac fibroblasts (DsRed-cell) were transplantedinto NOD-SCID mouse hearts 1 day after infection and sections of heartsanalyzed by immunocytochemistry after 2 weeks. Transplanted fibroblastsmarked with DsRed did not express actinin (green). (B) Cardiacfibroblasts infected with DsRed or Gata4/Mef2c/Tbx5 with DsRed(3F/DsRed-cell) were transplanted into NOD-SCID mouse hearts 1 day afterinfection. Note that a subset of 3F/DsRed cells (red) expressedα-MHC-GFP (green). Data were analyzed two weeks after transplantation.(C) Gata4/Mef2c/Tbx5-transduced cardiac fibroblasts (3F-cell) weretransplanted into mouse hearts. A subset of induced GFP⁺ cells expressedactinin (red) and had sarcomeric structures. Insets arehigh-magnification views (arrows). Data were analyzed two weeks aftertransplantation. Representative data are shown in each panel (n=4 ineach group). Scale bars, 100 μm.

Example 2 Direct Reprogramming of Cardiac Fibroblasts into FunctionalCardiomyocytes

Using methods essentially as described in Example 1, exogenous Gata4,Tbx5, and Mef2c were introduced into mouse post-natal tail tipfibroblasts. About 20% to 30% of the post-natal tail tip fibroblastswere reprogrammed to myosin heavy chain-GFP⁺ cells (cardiomyocytes)following introduction of Gata4, Tbx5, and Mef2c (where Gata4, Tbx5, andMef2c are collectively referred to as GMT).

Example 3 In Vivo Reprogramming of Murine Cardiac Fibroblasts intoCardiomyocytes

Materials and Methods

Retrovirus Generation, Concentration and Titration.

Retroviruses were generated as described in Example 1. The pMXsretroviral vectors containing coding regions of Gata4, Mef2c, Tbx5, anddsRed were transfected into Plat-E cells with Fugene 6 (Roche) togenerate viruses. Ultra-high titer virus (>1×10¹⁰ plaque-forming units(p.f.u) per ml) was obtained by standard ultracentrifugation. Retroviraltitration was performed using Retro-X qRT-PCR Titration Kit (Clontech).

Animals, Surgery, Echocardiography and Electrocardiography.

Postn-Cre; R26R-lacZ mice were obtained by crossing Periostin-Cre mice(Snider et al. (2009) Circulation Res. 105:934) and Rosa26-lacZ mice(Soriano (1999) Nat. Genet. 21:70). Postn-Cre; R26R-EYFP mice wereobtained by crossing Periostin-Cre mice and Rosa26-EYFP mice (Srinivaset al. (2001) supra). All surgeries and subsequent analyses wereperformed in a blinded fashion for genotype and intervention. Myocardialinfarction (MI) was induced by permanent ligation of the left anteriordescending artery (LAD) as described (Qian et al. (2011) J. Exp. Med.208:549). A pool of concentrated virus (GMT (Gata-4, Mef2c, Tbx5); orGMTR (GMT plus DsRed)) was mixed, and 10 μl of mixed virus plus 10 μl ofPBS or 40 ng/μl Thymosin β4 was injected along the boundary betweeninfarct zone and border zone (based on the blanched infarct area) aftercoronary artery occlusion. Mouse echocardiography and surfaceelectrocardiography were performed as described (Qian et al. (2011)supra).

Immunohistochemistry and Immunocytochemistry.

Immunohistochemistry and immunocytochemistry were performed as described(Qian et al. (2011) supra). Scar size was determined by Masson-Trichromestaining (Bock-Marquette et al. (2004) Nature 432:466; and Qian et al.(2011) supra). The area at risk (AAR) and myocardial infarct size weredetermined by Evans Blue/triphenyltetrazolium chloride labelingtechnique (Kurrelmeyer et al. (2000) Proc. Natl. Acad. Sci. USA97:5456).

Isolation of Adult Cardiomyocytes, Single Cell Patch Clamp, and CardiacFibroblast Migration Assay.

Adult cardiomyocyte isolation was performed as described with minormodifications (Xu et al. (1999) J. Gen. Physiol. 113:661). Single cellpatch clamp recordings were performed as described (Knollmann et al.(2003) Circulation Res. 92:428; Le Guennec et al. (1994) J. Physiol. 478Pt 3:493; Spencer et al. (2003) Am. J. Physiol. Heart Circ. Physiol.285:H2552). Migration assay was performed according to the publishedexplant culture protocol (Example 1; Bock-Marquette et al. (2004) supra;and Andersen et al. (2009) supra). In brief, isolated adult mouse heartswere minced into small pieces less than 1 mm³ in size. The explants wereplated on gelatin-coated dishes and cultured in explant medium (IMDM/20%FBS) until fibroblasts migrated out from minced tissue. The number ofdays when 10 heart pieces were identified with migratory fibroblasts wasrecorded.

FACS and Quantitative RT-PCR.

Dissociated cardiac cells from mouse hearts were stained withAPC-conjugated anti-Thy1 antibody (eBioscience) for 30 minutes at roomtemperature. After washing with PBS twice, stained cells were sorted byFACSAria2 (BD). RNA was extracted by TRizol method (Invitrogen). RT-PCRwas performed using the Superscript III first-strand synthesis system(Invitrogen). qPCR was performed using the ABI 7900HT (TaqMan, AppliedBiosystems), per the manufacturer's protocols. Optimized primers fromTaqman Gene Expression Array were used.

Statistics.

Differences between groups were examined for statistical significanceusing unpaired student's t-test or ANOVA. p<0.05 was regarded assignificant.

Results

Example 1 describes direct reprogramming of fibroblasts intocardiomyocyte-like cells in vitro upon expression of the threetranscription factors, Gata4, Mef2c, and Tbx5 (GMT). As observed inreprogramming to iPS cells, the percentage of fibroblast cells thatbecame fully reprogrammed to beating cardiomyocytes in vitro was small,but far more were partially reprogrammed, much like pre-iPS cells thathave potential to become fully pluripotent with additional stimuli. Itwas posited that cardiac fibroblasts may reprogram more fully in vivo intheir native environment, which might promote their survival,maturation, and coupling with neighboring cells. If so, the vastendogenous pool of cardiac fibroblasts could serve as a potential sourceof new cardiomyocytes for regenerative therapy.

To efficiently deliver GMT at high levels in vivo, a retroviral systemwas used to express GMT, or dsRed as a marker, into hearts of2-month-old male mice. 10 μl of ultra-high-titer retrovirus (˜10¹⁰copies/ml) that expressed each transcription factor and dsRed controlwas injected into the myocardial wall as a mixture. Two days afterretrovirus injection, transverse sections across the injected area wereprepared and co-stained for dsRed, the cardiomyocyte marker, α-Actinin,and Vimentin, which is enriched in the fibroblast population. While nomarkers are completely specific to cardiac fibroblasts, fibroblasts arecharacterized by expression of Vimentin and the surface markers Thy1 andDDR2 (Ieda et al. (2009) supra). At baseline, it was difficult to detectany α-Actinin- or Vimentin-positive cells that also expressed dsRed,suggesting minimal viral uptake. This was consistent with theobservation that retroviruses only infect cells that are activelydividing (Byun et al. (2000) J. Gene Med. 2:2).

Fibroblasts, which have an embryologic origin distinct fromcardiomyocytes, become activated after cardiac injury, such asmyocardial infarction (MI), and migrate to the site of injury andproliferate. Cardiac injury was induced by coronary artery ligation andinjected dsRed retrovirus into the myocardium bordering the infarctzone. While cells co-expressing dsRed and α-Actinin were stillundetectable, many Vimentin-positive cells, that were also positive fordsRed, were found (FIG. 7A). To quantify the percentage of heart cellsthat took up the virus, fluorescence activated cell sorting (FACS) wasused to analyze cells dissociated from the infarct/border zone ofinjected hearts two days after injury. Cells stained with Thy1, asurface marker enriched in fibroblasts, were increased upon surgery,suggesting successful activation of cardiac fibroblasts (FIG. 7B). RaredsRed⁺Thy1⁺cells were detected by FACS from sham-operated mice; however,4.2% of cells from the infarct/border zone of mice with MI weredsRed⁺Thy1⁺, suggesting successful delivery of virus into cardiacfibroblasts upon injury (FIG. 7C). In agreement with this, dsRed⁺Thy1⁺sorted cells expressed 60 fold higher levels of Gata4, Mef2c, and Tbx5than dsRed⁻Thy1⁺ cells by quantitative PCR (qPCR) (FIG. 7D).

To determine if cardiomyocyte conversion from a non-myocyte pool wasoccurring in vivo, lineage-tracing experiments were used to track theorigin of putative induced cardiomyocytes. Cardiac fibroblasts weregenetically labeled with a mouse line that expresses Cre-recombinaseunder the promoter of the fibroblast-enriched gene, Periostin (Snider etal. (2009) supra; and Snider et al. (2008) Circulation Res. 102:752).When intercrossed with the R26R-lacZ reporter line (Soriano et al.(1999) supra), which results in activation of β-galactosidase inPeriostin-expressing cells and all their progeny (FIG. 7E),β-galactosidase activity was found in the majority of cardiacfibroblasts and some endocardial and endothelial cells, as reported(Snider et al. (2009) supra; Snider et al. (2008) supra; and Takeda etal. (2010) J. Clin. Invest. 120:254). Most importantly, β-galactosidaseactivity was not detected in any cardiomyocytes, even after injury bycoronary ligation, in agreement with reports (FIG. 7F) (Snider et al.(2009) supra; Snider et al. (2008) supra; and Takeda et al. (2010)supra). However, 4 weeks after MI and retroviral delivery of GMT, manyβ-galactosidase⁺ cells that were also α-Actinin⁺ were found in theinjured area of the heart, suggesting that they may be descendants ofcells that once expressed Periostin (FIG. 7G). These cells hadwell-formed sarcomeres and were shaped similar to β-galactosidasemyocytes (FIG. 7G).

FIGS. 7A-G. Genetic Lineage Tracing Demonstrates In Vivo Reprogrammingof Cardiac Fibroblasts to Cardiomyocyte-Like Cells.

a, Confocal images of immunofluourescent staining on hearts showing theintegration of dsRed control virus (red) into Vimentin⁺ cells (green)but not into αActinin⁺ cells (blue) 2 days after MI. Arrows point todsRed⁺Vimentin⁺ cells (yellow), two of which were scanned under highmagnification and shown in the right three panels, with merged image atbottom. b, Quantification by FACS analyses of Thy-1 positive cells fromhearts 2 days after sham-operation or myocardial infarction (MI). n=3,*p<0.05. c, FACS analyses of Thy-1⁺dsRed⁺ cells from sham-operated or MImice with quantification (left) and representative FACS plots (right).n=3, *p<0.05. d, qPCR of Gata4, Mef2c and Tbx5 in Thy-1⁺dsRed⁺ cellscompared to Thy-1⁺dsRed⁻ cells sorted two days after GMTR (Gata4, Mef2c,Tbx5 and dsRed) was injected into hearts post-MI. n=3 with technicalquadruplicates. e, Schematic diagram showing the genetic fate mappingmethod to lineage trace cardiomyocyte-like cells reprogrammed fromPostn-Cre; R26R-lacZ cells. f, Immunofluorescent staining for αActinin(green), βGal (red), and DAPI (blue) on Postn-Cre; R26R-lacZ mouse heartsections 4 weeks after sham operation or MI. Note absence of αActinin⁺βGal⁺ double-positive cells even after MI. g, Immunofluorescent stainingfor αActinin (green), βGal (red), and DAPI (blue) on dsRed- orGMT-injected Postn-Cre; R26R-lacZ heart sections from border zone ofmice 4 weeks post MI. The lowest panels are magnified pictures of boxedareas in the middle panels.

The extent and spectrum to which the β-galactosidase⁺α-Actinin⁺ cellshad been reprogrammed was determined. To avoid the potential for falsepositive signals from overlaying cells due to the thickness of heartsections, adult cardiomyocytes were isolated at the single-cell levelfrom the infarct/border zone of Periostin-Cre; R26R-lacZ hearts 4 weeksafter coronary ligation and injection with GMT or dsRed control. Nocardiomyocytes isolated from dsRed-injected hearts were β-galactosidase⁺by immunostaining (FIG. 8A, n=6 animals, 4˜6 slides/animal); similarcells isolated from Periostin-Cre; R26R-EYFP mice showed no YFP⁺cardiomyocytes among thousands of cells visualized. In contrast, 35% ofcells isolated in the cardiomyocyte preparation from the border/infarctzone were β-galactosidase⁺ after GMT injection (FIGS. 8B and 8C). Amongthe β-galactosidase⁺ cells, 98% were α-Actinin+. To address thepossibility that the β-galactosidase⁺ cells might represent leakyexpression of Periostin-Cre or ectopic activation of Cre incardiomyocytes, hearts were co-injected with pooled retrovirus for GMTand dsRed (GMTR), providing a marker for non-myocyte cells that took upGMT retrovirus. It was found that the β-galactosidase⁺ cells were alsopositive for dsRed, indicating they were infected by virus, consistentwith their non-cardiomyocyte origin (FIGS. 8D-G).

Morphologically, the majority of β-galactosidase⁺ cells were large witha rod-shaped appearance and were binucleated, closely resemblingendogenous cardiomyocytes that were β-galactosidase negative from thesame isolation (FIGS. 8D-G). Further analyses revealed that, in additionto α-Actinin (FIG. 8H), the β-galactosidase⁺ cells expressed multiplesarcomeric markers, including Tropomyosin (FIG. 8I), cardiac muscleheavy chain (MHC) (FIG. 8J), and cardiac TroponinT (cTnT) (FIG. 8K).Examples of cells that showed nearly normal sarcomeric structuresthroughout the cell, representing ˜50% of cells, are shown (FIGS. 8H-K).The cells did not express Vimentin, smooth muscle α-actin, or SM22,suggesting the cells were no longer cardiac fibroblasts, nor did theybecome myofibroblasts or vascular smooth muscle cells. For simplicity,the β-galactosidase⁺α-Actinin⁺ cardiomyocyte-like cells are referred toas in vivo induced cardiomyocytes (iCMs), at least based on distinctivemorphology, gene expression and sarcomeric structure.

To determine if the iCMs expressed proteins involved in cell-cellcommunication with endogenous cardiomyocytes, the expression level andpattern of N-Cadherin, a cell-surface Ca²⁺-dependent adhesion moleculethat is located in intercalated disks in the myocardium, was examined.Over 90% of iCMs expressed N-Cadherin; 61% had localized N-Cadherin atthe cell border; and 5% fully resembled the endogenous cardiomyocytelocalization (FIG. 8L). Expression of Connexin 43 (Cx43), the major gapjunction protein in the heart that promotes electrical coupling of cellsand synchronized contraction of myocytes throughout the ventricle, wasalso examined. About 90% of iCMs expressed Cx43, with half of thoseexpressing Cx43 at high levels and in a pattern similar to endogenouscardiomyocytes with cell border localization (FIG. 8M); in 4%, Cx43 waslocalized in a pattern almost indistinguishable from that of anendogenous cardiomyocyte (FIG. 8M).

To determine if iCMs possessed the typical electrophysiologicalproperties of mature cardiomyocytes, intracellular electrical recordingwas performed by standard patch clamp techniques. Recording from asingle-cell suspension of cardiomyocytes from the border/infarct zone ofPeriostin-Cre; R26R-EYFP mice transduced with GMT, action potentials ofYFP⁺ cells (iCMs) and endogenous cardiomyocytes that were YFP⁻werecompared (FIG. 8N; and FIG. 9). Many reprogrammed cells had aphysiological resting membrane potential (−70 mV or less) and exhibitedcontraction in response to electrical stimulation but not at rest,similar to adult ventricular cardiomyocytes, which are normallyquiescent in the absence of stimulation. Varying action potentialmorphologies were identified (FIG. 8N; and FIG. 9), and somespontaneously contracting cells were observed, but these had restingpotentials around −50 mV. Taken together, many in vivo reprogrammedcardiomyocytes closely resembled adult ventricular cardiomyocytes 4weeks after introduction of GMT, while others were broadly similar,differing mainly in their ability to maintain a hyperpolarized restingpotential when evaluated as single cells in culture.

FIGS. 8A-N. Single-Cell Analysis of Cardiac Reprogramming In Vivo.

a-c, Immunofluorescent staining for βGal and DAPI on isolatedcardiomyocytes from the infarct/border zone of Postn-Cre; R26R-lacZhearts 4 weeks after dsRed (a) or GMT (b) injection with quantificationin (c). d-g, Bright-field image of CMs isolated from GMTR-injectedPostn-Cre; R26R-lacZ hearts 4 weeks after MI (d). Of these, (e) is βGalpositive and (f,g) are co-stained with dsRed. h-k, Immunofluorescentstaining for cardiac markers including αActinin, Tropomyosin, cardiacmyosin heavy chain (MHC), and cardiac Troponin T (cTnT) co-labeled withβGal and DAPI, on isolated cardiomyocytes from the infarct/border zoneof Postn-Cre; R26R-lacZ hearts 4 weeks after GMT injection. The picturesare representative examples of the induced cardiomyocytes next to anendogenous cardiomyocyte from the same preparation with quantificationand sample size. High magnification images of boxed areas are shown inthe far right panels. l-m, Immunofluorescent staining for N-Cadherin, orConnexin 43 co-labeled with βGal and DAPI, on isolated cardiomyocytesfrom the infarct/border zone of Postn-Cre; R26R-lacZ hearts 4 weeksafter GMT injection. Left two panels are representative images withquantification and sample size; right two panels represent examples ofthe best-reprogrammed induced cardiomyocytes next to an endogenouscardiomyocyte from the same preparation with quantification and samplesize. High magnification images of boxed areas are shown next to themerge pictures. n, Intracellular current clamp recording of multiplecardiomyocytes isolated from Postn-Cre; R26R-EYFP hearts 4 weeks afterMI. iCMs that were YFP⁺ displayed action potentials that resembled thoseof endogenous cardiomyocytes that were YFP⁻ from the same preparation.Cells were isolated from Postn-Cre; Rosa-EYFP mice 8 weeks post MI andvirus transduction.

FIG. 9. Intracellular recordings showing action potentials foradditional in vivo reprogrammed iCMs (YFP⁺ cells isolated fromPostn-Cre; Rosa-EYFP hearts 8 weeks after MI and infection with Gata4,Mef2c, and Tbx5).

Since in vivo reprogrammed iCMs had contractile potential and mayelectrically couple with viable endogenous cardiomyocytes, it wasdetermined if converting endogenous cardiac fibroblasts into myocytestranslates into partial restoration of heart function after MI. Allstudies were performed in blinded fashion, including retroviralinjection, and de-coded after completion of measurements. Mice injectedwith GMT or dsRed alone underwent serial high-resolutionechocardiography 1 day before and 3 days and 1, 4, 8, and 12 weeks afterMI. Using Evans blue/TTC double staining, the area at risk (AAR), andthe infarct size of myocardium 48 hours after coronary ligation, wereassessed. GMT- and dsRed-injected mice showed no differences in AAR orinfarct size (FIG. 10A), suggesting that the extent of initial cardiacinjury post-MI was not significantly affected by GMT induction. All miceshowed a reduction in left ventricular function after coronary arteryligation (FIG. 11; and FIG. 10B). 8 and 12 weeks after injection, thefraction of blood ejected from the left ventricle with each contraction(ejection fraction) and the fractional shortening of the ventricularchamber was significantly improved in mice injected with GMT, comparedto controls injected with dsRed (FIG. 11B; and FIGS. 10B-D). Strokevolume (volume of blood ejected with each heart beat) and cardiac outputwere improved after 8 weeks (FIG. 10C) and were close to normal after 12weeks (FIG. 11A).

As a molecular readout of cardiac dysfunction, qPCR was performed tomonitor the expression levels of atrial natriuretic factor (ANF), brainnatriuretic peptide (BNP) and tenascin C (Tnc) from GMT-injected andcontrol hearts in the area of injury. All were up-regulated after MI, asexpected, but this upregulation was attenuated by injection of GMT ininfarcted hearts (FIG. 11B). It was also found that the expression levelof collagen genes, which was increased upon MI, was partially restoredby injecting GMT (FIG. 11C). In agreement with the improvement ofcardiac function, injection of GMT resulted in a smaller scar size 8weeks after MI (FIG. 11D). ECG studies did not indicate evidence forarrhythmias with GMT injection compared to control dsRed injection, andno mice suffered sudden death.

FIGS. 10A-D. Determination of Area at Risk (AAR) and Infarct Size fordsRed or GMT Injected Hearts after Coronary Ligation and AdditionalEchocardiography Data.

a, Representative pictures of Evans blue TTC staining on four continuousslices of left ventricle from representative hearts of dsRed or GMTinjected hearts 48 hours after myocardial infarction (MI). Scale bars:500 p.m. Histogram is the blinded quantification of the area at risk(AAR) and infarct size as described in Methods. There was no statisticaldifference between dsRed and GMT injected MI hearts. b-c, Fractionalshortening (FS), ejection fraction (EF), stroke volume (SV), and cardiacoutput (CO) of the left ventricle are shown using high-resolutionechocardiography on hearts injected with dsRed or GMT. Changes in theseparameters 3 days (b) and 8 weeks (c) after MI are shown. d, Heart rateduring echocardiography is shown for each time point showing nodifference between dsRed and GMT cohorts. All echo data in (b, c, d)were collected in blinded fashion. dsRed, n=9; GMT, n=10. *p<0.05.

FIGS. 11A-D. In Vivo Delivery of Cardiac Reprogramming Factors ImprovesCardiac Function After Myocardial Infarction.

a, Fractional shortening (FS), ejection fraction (EF), stroke volume(SV), and cardiac output (CO) of the left ventricle were measured usinghigh-resolution echocardiography. Changes in these parameters before and12 weeks after MI were calculated. Data were collected in blindedfashion. dsRed, n=9; GMT (Gata4, Mef2c, Tbx5), n=10. *p<0.05. b, qPCR ofANF (Atrial Natriuretic Factor), BNP (Brain Natriuretic Peptide) and Tnc(Tenascin C) on RNA extracted from the border zone of hearts 4 weeksafter MI and injection of dsRed or GMT. c, qPCR of collagen type 1 alpha1 (Colla1), collagen type 1 alpha 2 (Colla2), collagen type III alpha1(Col3a1), elastin (Eln) on RNA extracted from the border zone of hearts4 weeks after MI and injection of dsRed or GMT. Data in (b) and (c) areshown relative to dsRed injected sham-operated mice, indicated by dashedline. n=3 for each genotype with technical quadruplicates. d,Masson-Trichrome staining of heart sections 8 weeks post-MI injectedwith dsRed or GMT. Scale bars: 500 μm. Quantification of scar size wascalculated by measurement of scar area in four sections for each heartin blinded fashion. dsRed, n=8; GMT, n=9. *p<0.05.

While GMT delivery significantly affected cardiac repair after MI, itwas hypothesized that increasing the number of Thy1⁺ cells that wereinfected by the retrovirus might lead to an even greater functionalimprovement. Thymosin β4, a 43-amino-acid G-actin monomer-bindingprotein, promotes cell migration, cardiac cell survival and can activateepicardial cells to become more proliferative and give rise to morecardiac fibroblasts and endothelial cells. It was previously reportedthat Thymosin β4 improves cardiac function and decreases scar size afterMI. To test the effects of Thymosin β4 on cardiac fibroblast migration,a cardiac explant migration assay was used. The average time forfibroblasts to migrate from adult heart explants was approximately 3weeks; however, with Thymosin β4, equivalent fibroblast migration wasobserved after only 2 weeks and was even more accelerated after MI (FIG.12A). Similarly, the proliferation of Vimentin⁺ cells increased afterMI, and increased even further with Thymosin β4, as marked byphosphohistone H3 (FIG. 12B). Consistent with the activation offibroblasts by Thymosin β4, the percent of Thy1⁺ (FIG. 12C) or Vimentin⁺(FIG. 13A) cells infected by retrovirus in the setting of MI more thandoubled upon intramyocardial injection of Thymosin β4 (FIG. 12C). Theimproved delivery of GMT-expressing retrovirus by Thymosin β4 resultedin an increase in the percent of βGal⁺ iCMs, compared to totalcardiomyocytes, in single-cell cardiomyocyte culture from theinfarct/border zone of Periostin-Cre; R26R-lacZ hearts (51% vs. 35%,p<0.05) (FIG. 12D). However, no change was observed in the in vivoreprogramming efficiency (iCMs/total cells infected with GMT virus),which remained ˜12% (FIG. 12D), or the degree of reprogramming (FIG.13B).

Injection of Thymosin β4 immediately after coronary ligation resulted infunctional improvement of cardiac function, as reported. Co-injection ofThymosin β4 and GMT yielded further functional improvement in ejectionfraction and cardiac output 8 weeks after infarction (FIG. 13E; andFIGS. 12C and 12D). In agreement with this, co-injection of Thymosin β4and GMT caused a greater reduction in scar size than either Thymosin β4or GMT injection alone (FIG. 12F), despite the area at risk and initialinfarct size being similar in both groups (FIG. 13E).

FIGS. 12A-F. Thymosin β4 activates cardiac fibroblasts upon injury andenhances in vivo reprogramming. a, Quantification for cardiac fibroblastmigration assay performed on sham-operated or post-MI hearts with orwithout Tβ4 injection. Days when 10 minced cardiac tissues weresurrounded by migratory fibroblasts (“islands”) were averaged from threeinjected hearts. b, Immunofluorescent staining for phosphohistone H3(pH3, red), Vimentin (green) and DAPI (blue) on heart sections 48 hoursafter sham-operation, MI or MI with injection of Tβ4. At right,quantification for pH3⁺Vimentin⁺ cells. n=3 for each genotype. c, FACSanalyses of Thy-1⁺dsRed⁺ cells from hearts 2 days after sham-operation,MI, or MI with injection of Tβ4 with quantification (left) andrepresentative FACS plots (right). n=3. d, Upper panels showimmunofluorescent staining for βGal and DAPI on isolated CMs frominfarct/border zone of Postn-Cre; R26R-lacZ hearts 4 weeks after MI andGMT injection with or without Tβ4. Lower panels are bright fieldpictures for the same cells. Quantification of βGal⁺ cardiomyocyte(CM)-like cells compared to total CMs or total dsRed⁺ cells(virus-infected) from the border zone of hearts 4 weeks after MI andinjection of GMT with or without Tβ4. n=3. e, Changes in ejectionfraction (EF) and cardiac output (CO) of the left ventricle weredetermined using high-resolution echocardiography 8 weeks post-surgery.dsRed, n=9; GMT, n=10; dsRed+Tβ4, n=10; GMT+Tβ4, n=8. f, Scar areacalculated in blinded fashion from multiple heart sections 8 weekspost-MI after dsRed (n=8), GMT (n=9), dsRed+Tβ4 (n=7) or GMT+Tβ4 (n=8)injection. Representative Masson-Trichrome staining on heart sections isshown. Scale bars: 500 p.m. Quantification of scar size was calculatedby measurement of scar area in blinded fashion. *p<0.05; **p<0.01.

FIGS. 13A-E. Thymosin β4 Activates Cardiac Fibroblasts Upon Injury andEnhances In Vivo Reprogramming.

a, Confocal images showing the integration of dsRed control virus intoVimentin⁺ cells in hearts with or without Thymosin β4 (Tβ4) injectionafter myocardial infarction (MI). Arrows point to dsRed⁺Vimentin⁺ cells.b, Quantification of iCM phenotypes after single cell isolation fromhearts injected with Tβ4 and GMT. c, Blinded quantification of the areaat risk (AAR) and infarct size of hearts co-injected with Thymosin β4and dsRed or GMT 48 hours after MI. d, Changes in fractional shortening(FS) and stroke volume (SV) of the left ventricle 8 weeks after MI usinghigh-resolution echocardiography. dsRed, n=9; GMT, n=10; dsRed+Tβ4,n=10; GMT+Tβ4, n=8. *p<0.05, **p<0.01. e, Heart rate was measured duringechocardiography and no difference was found among the four groups. Echodata in (d, e) were collected in blinded fashion.

The above-described experiments show that resident cardiac fibroblastscan be converted into cardiomyocyte-like cells in vivo by local deliveryof Gata4, Mef2c, and Tbx5 using retroviral-mediated gene transfer uponcardiac injury. Compared to in vitro conversion, in vivo cardiacreprogramming occurred with similar initial efficiency. Reprogrammedcells more closely resembled endogenous cardiomyocytes and were morefully reprogrammed.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. An in vivo method of generating an induced cardiomyocyte, the method consisting of: genetically modifying a rodent post-natal fibroblast with one or more nucleic acids encoding only three reprogramming factor polypeptides, wherein the three reprogramming factor polypeptides consist of Gata4, Mef2c, and Tbx5, and wherein said genetic modification results in direct reprogramming of the rodent post-natal fibroblast into a cardiomyocyte, thereby generating an induced cardiomyocyte.
 2. The method of claim 1, wherein said one or more nucleic acids is a recombinant vector.
 3. The method of claim 1, wherein said one or more nucleic acids are operably linked to a transcription regulatory element.
 4. The method of claim 3, wherein the transcription regulatory element is a constitutive promoter functional in the post-natal fibroblast.
 5. An in vivo method of generating an induced cardiomyocyte, the method consisting of: genetically modifying a rodent post-natal fibroblast with one or more nucleic acids encoding only three reprogramming factor polypeptides, wherein the three reprogramming factor polypeptides consist of Gata4, Mef2c, and Tbx5, and wherein said genetic modification results in direct reprogramming of the rodent post-natal fibroblast into a cardiomyocyte, thereby generating an induced cardiomyocyte; and isolating the induced cardiomyocyte.
 6. The method of claim 1, wherein the three reprogramming factor polypeptides are encoded by a single nucleic acid.
 7. The method of claim 5, wherein the three reprogramming factor polypeptides are encoded by a single nucleic acid. 